Lumazine synthase and riboflavin synthase

ABSTRACT

Through function complementation of E. coli auxotrophs, the ultimate and pentultimate enzymes of the spinach riboflavin biosynthetic pathway have been cloned, namely, lumazine synthase (LS) and riboflavin synthase (RS). This invention relates to the isolation of nucleic acid fragments from plants or fungi that encode LS protein. The invention also relates to the isolation of nucleic acid fragments from plants or fungi that encode RS protein. In addition, the invention also relates to the construction of chimeric genes encoding all of a portion of LS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant LS in a transformed host cell. Furthermore, the invention also relates to the construction of chimeric genes encoding all of a portion of RS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant or fingal RS in a transformed host cell. In vivo and in vitro methods to identify herbicide or fungicide candidates are included that evaluate the ability of a chemical compound to inhibit the activity of a plant or fungal LS enzyme or a plant or fungal RS enzyme.

This is a continuation of Ser. No. 08/912,218 filed Aug. 15, 1997.

FIELD OF THE INVENTION

This invention is in the field of plant and fungal molecular biology.More specifically, this invention pertains to nucleic acid fragmentsencoding proteins involved in the riboflavin biosynthetic pathway ofplants or fungi.

BACKGROUND OF THE INVENTION

Riboflavin, vitamin B₂, is the precursor of flavin mononucleotide (FMN)and flavin adenine dinucleotide (FAD), essential cofactors for a numberof mainstream metabolic enzymes that mediate hydride, oxygen, andelectron transfer reactions. Riboflavin-dependent enzymes includesuccinate dehydrogenase, NADH dehydrogenase, ferredoxin-NADP⁺oxidoreductase, acyl-CoA dehydrogenase, and the pyruvate dehydrogenasecomplex. Consequently, fatty acid oxidation, the TCA cycle,mitochondrial electron-transport, photosynthesis, and numerous othercellular processes are critically dependent on either FMN or FAD asprosthetic groups. Other notable flavoproteins include glutathionereductase, glycolate oxidase, P450 oxido-reductase, squalene epoxidase,dihydroorotate dehydrogenase, and α-glycerophosphate dehydrogenase.Genetic disruption of riboflavin biosynthesis in E. coli (Richter etal., J Bacteriol. 174:4050-4056 (1992)) and S. cerevisiae (Santos etal., J. Biol. Chem. 270:437-444 (1995)) results in a lethal phenotypethat is only overcome by riboflavin supplementation. This is notsurprising, considering the ensemble of deleterious pleiotropic effectsthat would occur with riboflavin deprivation.

Riboflavin is synthesized by plants and numerous microorganisms,including bacteria and fungi (Bacher, A., Chemistry and Biochemistry ofFlavoproteins (Muller, F., ed.) vol. 1, pp. 215-259, Chemical RubberCo., Boca Raton, Fla. (1990)). Since birds, mammals, and other higherorganisms are unable to synthesize the vitamin and, instead, rely on itsdietary ingestion to meet their metabolic needs, the enzymes that areresponsible for riboflavin biosynthesis are potential targets for futureantibiotics, fungicides, and herbicides. Moreover, it is possible thatthe distantly-related plant and microbial enzymes have distinctcharacteristics that could be exploited in the development of potentorganismspecific inhibitors. Thus, a detailed understanding of thestructure, mechanism, kinetics, and substrate-binding properties of theriboflavin biosynthetic enzyme(s), from plants for example, would serveas a starting point for the rational design of chemical compounds thatmight be useful as herbicides. Having the authentic plant protein(s) inhand Would also provide a valuable tool for the in vitro screening ofchemical libraries in search of riboflavin biosynthesis inhibitors.

Bacterial and fungal riboflavin biosynthesis has been intensivelystudied for more than four decades (For recent reviews, see Bacher, A.Chemistry and Biochemistry of Flavoproteins (Muller, F., ed.) vol. 1,pp. 215-259 and 293-316 Chemical Rubber Co., Boca Raton, Fla. (1990)).The synthetic pathway consists of seven distinct enzyme catalyzedreactions, with guanosine 5'-triphosphate (GTP) and ribulose 5-phosphatethe ultimate precursors. While the second and third steps of riboflavinbiosynthesis occur in opposite order in bacteria and fungi, theremaining pathway intermediates are identical in both microorganisms.Structurally and mechanistically, the last two reactions in the pathway,namely, those catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (LS)and riboflavin synthase (RS), are best characterized. In B. subtilis,these two enzymes are physically associated with each other in a hugespherical particle with a combined molecular mass of about 1 MDa (Bacheret al., J Biol Chem. 255:632-637 (1980); Ritsert et al., J. Mol. Biol.253, 151-167 (1995); Bacher et al., Biochem. Soc. Trans. 24(1):89-94(1996)); the X-ray structure of the bifunctional protein complex hasbeen determined at 3.3 angstrom resolution (Ladenstein et al., J. Mol.Biol 203:1045-1070). The LS/RS complex consists of 60 LS subunits thatare organized into 12 pentamers to form a hollow icosahedral capsid.Encaged in the central core of this structure resides a single moleculeof RS, a trimer of three identical subunits. Kinetic studies reveal thatthe compartmentation of the two enzymes within the complex improves theoverall catalytic efficiency of riboflavin production at low substrateconcentrations, presumably via "substrate channeling" (Kis et al., J.Biol. Chem. 270:16788-16795 (1995)). Although a bifunctional LS/RScomplex has not been observed in other microorganisms, it was recentlyshown that the native E. coli LS also exists in vivo as a hollowicosahedral capsid of 60 identical subunits (Mortl et al., J. Biol Chem.271:33201-033207 (1996)).

LS, the penultimate enzyme of riboflavin biosynthesis, catalyzes thecondensation of 3,4-dihydroxy-2-butanone 4-phosphate with4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) to yield 1 moleach of orthophosphate and 6,7-dimethyl-8-(1'-D-ribityl)-lumazine(DMRL). The latter is the immediate precursor of riboflavin. LS-encodinggenes have been cloned from numerous microorganisms, including E. coli(Taura et al., Mol. Gen. Genet. 234:429-432 (1992)), A. pleuropneumoniae(Fuller et al., J. Bacteriol. 177:7265-7270 (1995)), P. phosphoreum (Leeet al., J. Bacteriol. 176:2100-2104 (1994)), B. subtilis (Mironov etal., Dokl. Akad. Nauk SSSR 305:482-487 (1989)), and S. cerevisiae(Garcia-Ramirez et al., J. Biol Chem. 270:23801-23807 (1995)). In allcases, the subunit molecular mass of the LS gene product is small,ranging in size from ˜16-17 kDa. While the various LS homologs all sharecertain structural features in common, their overall homology at theprimary amino acid sequence level is rather poor. For example, asdetermined with the Genetics Computer Group Gap program (WisconsinPackage Version 9.0. Genetics Computer Group (GCG), Madison, Wis.), theE. coli LS is only 58%, 65%, 53%, and 36% identical to the homologousproteins of A. pleuropneumoniae, P. phosphoreum, B. subtilis and S.cerevisiae, respectively. Indeed, pair wise comparisons of these fiveproteins reveal that the two most similar homologs share only 72%identity.

The terminal step of riboflavin biosynthesis is mediated by RS. Thisenzyme catalyzes the dismutation of two molecules of DMRL to yield I molof riboflavin and RAADP. That the latter product is also one of thesubstrates of LS explains in part the enhanced catalytic efficiency ofthe B. subtilis LS/RS complex noted above. Although the crystalstructure of RS remains to be determined, it is surmised that the nativebacterial (Bacher et al., J. Biol Chem. 255:632-637 (1980)) and fungal(Santos et al., J. Biol. Chem. 270:437-444 (1995)) proteins are trimers,each consisting of three identical ˜25 kDa subunits. To date, RS hasonly been cloned from about a dozen microorganisms, and all of thespecies that have been examined exhibit marked internal homology intheir N-terminal and C-terminal domains (Schott et al., J. Biol. Chem.265:4204-4209 (1990); Santos et al., J. Biol Chem. 270:437-444 (1995)).Based on these observations, it has been suggested that the two halvesof the RS protomer have arisen through gene duplication, and that eachcontains a substrate-binding site for DMRL. Despite this structuralsimilarity, however, the overall sequence homology of the various RSproteins is extremely limited. Thus, the E. coli RS protein is only 32%,36%, 35%, and 31% identical to its counterparts in S. cerevisiae, P.phosphoreum., B. subtilis, and P. leiognathi; the GenBank accessionnumbers for the latter four proteins are Z21621, L11391, X51510 andM90094, respectively.

With the exception of GTP cyclohydrolase II, the first committed enzymeof riboflavin biosynthesis, virtually nothing is known about theriboflavin biosynthetic machinery of higher plants. The gene for thisprotein was recently cloned from an arabidopsis cDNA library (Kobayashiet al., Gene 160:303-304 (1995)). The protein sequence of the clonedplant gene is only 37-58% identical to the homologous proteins from E.coli, B. subtilis, P. leiognathi, and P. phosporeum. While full-lengthcDNA sequences have not been reported for any other plant riboflavinbiosynthetic enzyme, the GenBank database contains two ESTs (ExpressedSequence Tags) that potentially correspond to plant LS genes. One ofthese is from castor bean and the other is from arabidopsis.

The castor bean cDNA clone (GenBank accession number T15152; van de Looet al. Plant Physiol. 108:1141-1150 (1995)) is truncated at its 5' end,and is missing DNA corresponding to at least 60 N-terminal amino acidresidues. The arabidopsis cDNA clone (GenBank accession number Z34233;direct submission) was identified through a BLAST (Basic Local AlignmentSearch Tool; Altschul et al. J. Mol. Biol. 215:403-410 (1990)) searchusing the TBLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The query sequence for the BLASTsearch was the translated E. coli LS gene (GenBank accession numberX64395) and the probability score for similarity to the arabidopsis ESTwas P=0.45. Unfortunately, the portion of the cDNA insert that wassequenced contained only the last 26 C-terminal amino acid residues ofthe protein, so it is not known whether it is a partial or full-lengthcDNA clone. Since neither of these clones possess a polyA tail, it ispossible that they reflect contaminating microbial DNA that wasintroduced at some point during the preparation of the cDNA libraries.

In contrast to LS, BLAST searches failed to identify any plant DNAsequences in the GenBank database with significant primary amino acidsequence homology to either E. coli or yeast RS. However, RS activityhas been detected in extracts from various plant species (Plaut, G.,Metabolic Pathways (Greenberg, D. M., ed.), vol II, p. 673, AcademicPress, New York, (1961)), and partial purification of the spinachhomolog has been described (Mitsuda et al., Methods Enzymol. 18b:539-543(1970)).

From the foregoing discussion, it is apparent that too little is knownabout plant LS or RS genes/proteins and their relationship to knownmicrobial homologs to allow isolation of LS- or RS-encoding genes fromany plant species using most classical approaches. The latter includehybridization probing of cDNA libraries with homologous or heterologousgenes, PCR-amplification of the gene of interest using oligionucleotideprimers corresponding to conserved amino acid sequence motifs, and/orimmunological detection of expressed cDNA inserts in microbial hosts.Unfortunately, these techniques would not be expected to be very usefulfor the isolation of plant LS or RS genes, since they all heavily relyon the presence of significant structural similarity (i.e., DNA or aminoacid sequence) with known proteins and genes that have the samefunction. Given the observation that LS and RS proteins are both sopoorly conserved, even amongst microorganisms, it is highly unlikelythat the known microbial homologs would share significant structuralsimilarities with their counterparts in higher plants.

An alternative approach that has been used to clone biosynthetic genesin other metabolic pathways from higher eucaryotes is throughcomplementation of microbial mutants that are deficient in the enzymeactivity of interest. Since this strategy relies only on the functionalsimilarity between the protein encoded for by the disrupted host geneand the target gene of interest, it is ideally suited for cloningstructurally dissimilar proteins that catalyze the same reaction. Forfunctional complementation, a cDNA library is constructed in a vectorthat can direct the expression of the cDNA in the microbial host. Theplasmid library is then introduced into the mutant microbe, and coloniesare selected that are no longer phenotypically mutant. Indeed, the LS(Garcia-Ramirez et al., J. Biol. Chem. 270:23801-23807 (1995)) and RS(Santos et al., J. Biol. Chem. 270:437-444 (1995) of yeast, andarabidopsis GTP cyclohydrolase II (Kobayashi et al, Gene 160:303-304(1995)) were all cloned through functional complementation of microbialriboflavin auxotrophs. This strategy has also worked for isolating genesfrom higher eucaryotes that are involved in other metabolic pathways,including lysine biosynthesis (Frisch et al., Mol. Gen. Genet.228:287-293 (1991)), purine biosynthesis (Aimi et al., J. Biol. Chem.265:9011-9014 (1990)), and tryptophan biosynthesis (Niyogi et al., PlantCell 5:1011-1027 (1993)), and has also been successfully employed in theisolation of various plant genes including glutamine synthetase (Snustadet al., Genetics 120:1111-1124 (1988)), pyrroline-5-carboxylatereductase (Delauney et al., Mol. Genet. 221:299-305 (1990)),dihydrodipicolinate synthase (Frisch et al., Mol. Gen. Genet.228:287-293 (1991)), 3-isopropylmalate dehydrogenase (Ellerstrom et al.,Plant Mol. Biol. 18:557-566 (1992)), and dihydroorotate dehydrogenase(Minet et al., Plant J. 2:417-422 (1992)).

Despite the obvious attractive features of cloning by functionalcomplementation, there are several reasons why this approach might notwork when applied to the higher plant LS and RS genes. First, theeucaryotic cDNA sequence might not be expressed at adequate levels inthe mutant microbe for a variety of reasons, including differences inpreferred codon usage. Second, the cloned eucaryotic gene might notproduce a functional polypeptide, if for instance, enzyme activityrequires a post-translational modification, such as acetylation,glycosylation, or phosphorylation that is not carried out by themicrobial host. Third, the heterologous plant protein might be lethal tothe host, thus rendering its expression impossible. Fourth, theeucaryotic protein might fail to achieve its native conformation in theforeign microbial environment, due to folding problems, inclusion bodyformation, or various other reasons. It is also possible that the higherplant LS and RS enzymes are nuclear-encoded proteins that areposttranslationally targeted to chloroplasts, mitochondrial, or someother organelle that is not present in the microbial host. If this werethe case and proteolytic removal of the organellar targeting sequencewas required for enzyme activity, cloning these genes by functionalcomplementation would not be possible.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding plant or fungal enzymes involved in riboflavin biosynthesis.Specifically, this invention concerns isolated nucleic acid fragmentsencoding a plant or fungal LS, wherein the plant is spinach, tobacco orarabidopsis and the fungus is Magnaporthe grisea. This invention alsoconcerns isolated nucleic acid fragments encoding a plant or fungal RS,wherein the plant is spinach or arabidopsis and the fungus isMagnaporthe grisea. In addition, this invention relates to nucleic acidfragments that are complementary to nucleic acid fragments encoding aplant or fungal LS enzyme or a plant or fungal RS enzyme.

Specific isolated nucleic acid fragments encoding a plant LS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; (b) an isolated nucleic acidfragment that is substantially similar to an isolated nucleic acidfragment encoding all or a substantial portion of the amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4and SEQ ID NO:6; (c) an isolated nucleic acid fragment encoding apolypeptide having at least 72% identity with the amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:6; and (d) an isolated nucleic acid fragment that is complementaryto (a), (b) or (c).

Special isolated nucleic acid fragments encoding a plant RS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence selected from the group consisting ofSEQ ID NO:8 and SEQ ID NO: 10; (b) an isolated nucleic acid fragmentthat is substantially similar to an isolated nucleic acid fragmentencoding all or a substantial portion of the amino acid sequenceselected from the group consisting of SEQ ID NO:8 and SEQ ID NO: 10; (c)an isolated nucleic acid fragment encoding a polypeptide having at least70% identity with the amino acid sequence selected from the groupconsisting of SEQ ID NO:8 and SEQ ID NO: 10; and (d) an isolated nucleicacid fragment that is complementary to (a), (b) or (c).

Specific isolated nucleic acid fragments encoding a fungal RS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence set forth in SEQ ID NO: 12; (b) anisolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding all or is homologous to at leasta substantial portion of the amino acid sequence set forth in SEQ IDNO:12: (c) an isolated nucleic acid fragment that is complementary to(a) or (b).

Specific isolated nude acid fragments encoding a fungal LS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence set forth in SEQ ID NO:38; (b) anisolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding all or is homologous to at leasta substantial portion of the amino acid sequence set forth in SEQ IDNO:38; and (c) an isolated nucleic acid fragment that is complementaryto (a) or (b).

In another embodiment, the instant invention relates to chimeric genesencoding a plant or fungal LS enzyme or a plant or fungal RS enzyme orto chimeric genes that comprise nucleic acid fragments that arecomplementary to the nucleic acid fragments encoding the enzymes,operably linked to suitable regulatory sequences, wherein expression ofthe chimeric genes results in production of levels of the encodedenzymes in transformed host cells that are altered (i.e., increased ordecreased) from the levels produced in the untransformed host cells.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a plant orfungal LS enzyme or a plant or fungal RS enzyme, operably linked tosuitable regulatory sequences, wherein expression of the chimeric generesults in production of altered levels of a plant LS enzyme or a plantor fungal RS enzyme in the transformed host cell. The transformed hostcells can be of eucaryotic or procaryotic origin, and include cellsderived from higher plants and microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and from seeds derived from such transformed plants.

An additional embodiment of the instant invention, concerns a method ofaltering the level of expression of a plant or fungal LS enzyme or aplant or fungal RS enzyme in a transformed host cell comprising: a)transforming a host cell with the chimeric gene encoding a plant orfungal LS enzyme or a plant or fungal RS enzyme, operably linked tosuitable regulatory sequences; and b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of alteredlevels of LS or RS in the transformed cell.

An additional embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or substantially all ofan amino acid sequence encoding a plant or fungal LS enzyme or a plantor fungal RS enzyme.

Additionally, an in vivo method is provided for identifying as anherbicidal or fungicidal candidate a chemical compound that inhibits theactivity of a plant or fingal LS enzyme or a plant or fungal RS enzymeand thus serve as a crop protection chemical comprising the steps of:(a) disrupting the endogenous LS or RS gene of a suitable microbialhost, rendering growth of the microbial host cell dependent on addedriboflavin; (b) transforming the altered microbial host cell of step (a)with a chimeric gene comprising an isolated nucleic acid fragmentencoding a plant or fungal LS enzyme or a plant or fungal RS enzyme, thechimeric gene operably linked to at least one suitable regulatorysequence that, allows its expression in the microbial host cell; (c)growing the transformed host cell of step (a) under conditions suitablefor expression of the chimeric gene plant or fungal LS or RS gene; (d)contacting the transformed microbial host cell with a chemical compoundof interest in a well-controlled experiment while the host cell isgrowing exponentially and in both the presence and absence of addedriboflavin; (e) identifying as an herbicide or fungicide candidate thechemical compound of interest that inhibits growth of the transformedmicrobial host cell only when grown in the absence of added riboflavin.Suitable isolated nucleic acid fragments are those set out above.Suitable microbial hosts for this in vivo assay include the E. coli LSand RS riboflavin auxotrophs that are described below, both of whichnormally require riboflavin supplementation for growth. Specificinhibition of the functional plant or fungal genes that are introducedinto these mutants could then be assessed directly in parallel assays inwhich the transformed host cells are grown in both the presence andabsence of added riboflavin. Those inhibitory compounds that only affectmetabolic activity (growth) in the absence of riboflavin supplementationrepresent potential herbicides and/or fungicides.

In an alternate embodiment, an in vitro method is provided foridentifying as an herbicide or fungicide candidate a chemical compoundthat inhibits the activity of a plant or fungal LS enzyme or a plant orfungal RS enzyme and thus serve as a crop protection chemical comprisingthe steps of: (a) transforming a host cell with a chimeric genecomprising a nucleic acid fragment encoding a plant or fungal LS enzymeor a plant or fungal RS enzyme, the chimeric gene operably linked to atleast one suitable regulatory sequence; (b) growing the transformed hostcell of step (a) under conditions suitable for expression of thechimeric gene resulting in the production of the plant or fungal LSenzyme or a plant or fungal RS enzyme; (c) purifying the plant or fungalLS enzyme or the plant or fungal RS enzyme expressed by the transformedhost cell; (d) contacting the enzyme with a chemical compound ofinterest; and (e) identifying as an herbicide or fungicide candidate thechemical compound of interest the activity of the plant or fungal LSenzyme or plant or fungal RS enzyme relative to the activity of therespective enzyme in the absence of the chemical compound of interest.Such reduced activity indicates that the chemical compound ispotentially useful as a crop protection chemical. Suitable isolatednucleic acid fragments are those set out above.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIGS. 1-4 show primary amino acid sequence alignments generated with theGCG Pile up program (Genetics Computer Group, Madison, Wis.).

FIG. 1 shows the primary amino acid sequence alignments of knownmicrobial RS homologs and the cloned spinach RS precursor protein.

FIG. 2 shows the primary amino acid sequence alignments of knownmicrobial LS homologs and the cloned spinach LS precursor protein.

FIG. 3 shows the primary amino acid sequence alignments of spinach andarabidopsis RS precursor proteins. Boxed residues denote the putativechloroplast targeting sequences (e.g., transit peptides).

FIG. 4 shows primary amino acid sequence alignments of spinach, tobaccoand arabidopsis LS precursor proteins. Boxed residues denote theputative chloroplast targeting sequences (e.g., transit peptides).

The following sequence descriptions and sequence listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219(2):345-373 (1984) which are herein incorporated byreference. The present invention utilized Wisconsin Package Version 9.0software from Genetics Computer Group (GCG), Madison, Wis.

SEQ ID NO:1 is the nucleotide sequence of a cloned cDNA encoding amature spinach LS.

SEQ ID NO:2 is the deduced amino acid sequence of the cloned cDNAencoding a mature spinach LS.

SEQ ID NO:3 is the nucleotide sequence of a cloned cDNA encoding amature tobacco LS.

SEQ ID NO:4 is the deduced amino acid sequence of the cloned cDNAencoding a mature tobacco LS.

SEQ ID NO:5 is the nucleotide sequence of a cloned cDNA encoding amature arabidopsis LS.

SEQ ID NO:6 is the deduced amino acid sequence of the cloned cDNAencoding a mature arabidopsis LS.

SEQ ID NO:7 is the nucleotide sequence of a cloned cDNA encoding amature spinach RS.

SEQ ID NO:8 is the deduced amino acid sequence of the cloned cDNAencoding a mature spinach RS.

SEQ ID NO:9 is the nucleotide sequence of a cloned cDNA encoding amature arabidopsis RS.

SEQ ID NO: 10 is the deduced amino acid sequence of the cloned cDNAencoding a mature arabidopsis RS.

SEQ ID NO: 11 is the nucleotide sequence of a cloned cDNA encoding aMagnaporthe grisea RS.

SEQ ID NO:12 is the deduced amino acid sequence of the cloned cDNAencoding Magnaporthe grisea RS.

SEQ ID NO:13 is the 5' primer useful in the amplification of E. coli LShaving Genbank accession No. X64395.

SEQ ID NO:14 is the 3' primer useful in the amplification of E. coli LShaving Genbank accession No. X64395.

SEQ ID NO:15 is the 5' primer useful in the amplification of E. coli RShaving Genbank accession No. X69109.

SEQ ID NO:16 is the 3' primer useful in the amplification of E. coli RShaving Genbank accession No. X69109.

SEQ ID NO:17 is the 5' primer useful for the introduction of a DNAfragment that confers kanamycin resistance into the E. coli LS and RSgenes having Genbank accession Nos. X64395 and X69109, respectively, ata Not1 cleavage site.

SEQ ID NO:18 is the 3' primer useful for the introduction of a DNAfragment that confers kanamycin resistance into E. coli LS and RS geneshaving Genbank accession Nos. X64395 and X69109, respectively, at a Not1cleavage site.

SEQ ID NO:19 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli LS having Genbank accessionNo. X64395 (hybridizes to nt 2273-2290).

SEQ ID NO:20 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli LS having Genbank accessionNo. X64395 (hybridizes to nt 2243-4261).

SEQ ID NO:21 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli RS having Genbank accessionNo. X69109 (hybridizes to nt 1217-1233).

SEQ ID NO:22 is the one of the PCR primers useful for the introductionof a NotI cleavage site in the middle of E. coli RS having Genbankaccession No. X69109 (hybridizes to nt 1190-1208).

SEQ ID NO:23 is the 5' primer useful for the removal of the transitpeptide from the cloned spinach RS precursor.

SEQ ID NO:24 is the 3' primer useful for the removal of the transitpeptide from the cloned spinach RS precursor.

SEQ ID NO:25 is the 5' primer useful for the removal of the transitpeptide from the cloned spinach LS precursor.

SEQ ID NO:26 is the 3' primer useful for the removal of the transitpeptide from the cloned spinach LS precursor.

SEQ ID NO:27 is the nucleotide sequence of a cloned cDNA encoding aspinach LS precursor with its transit peptide.

SEQ ID NO:28 is the deduced amino acid sequence of the cloned CDNAencoding a spinach LS precursor with its transit peptide.

SEQ ID NO:29 is the nucleotide sequence of a cloned cDNA encoding atobacco LS precursor with its transit peptide.

SEQ ID NO:30 is the deduced amino acid sequence of the cloned cDNAencoding a tobacco LS precursor with its transit peptide.

SEQ ID NO:31 is the nucleotide sequence of a cloned cDNA encoding anarabidopsis LS precursor with its transit peptide.

SEQ ID NO:32 is the deduced amino acid sequence of the cloned cDNAencoding an arabidopsis LS precursor with its transit peptide.

SEQ ID NO:33 is the nucleotide sequence of a cloned cDNA encoding aspinach RS precursor with its transit peptide.

SEQ ID NO:34 is the deduced amino acid sequence of the cloned cDNAencoding a spinach RS precursor with its transit peptide.

SEQ ID NO:35 is the nucleotide sequence of a cloned cDNA encoding anarabidopsis RS precursor with its transit peptide.

SEQ ID NO:36 is the deduced amino acid sequence of the cloned cDNAencoding an arabidopsis RS precursor with its transit peptide.

SEQ ID NO:37 is the nucleotide sequence of a cloned cDNA encoding aMagnaporthe grisea LS.

SEQ ID NO:38 is the deduced amino acid sequence of the cloned cDNAencoding Magnaporthe grisea LS.

SEQ ID NO:39 is the highly conserved C-terminal amino acid sequencefound in plant LS proteins.

DETAILED DESCRIPTION OF THE INVENTION

Luminase synthase (LS) and riboflavin synthase (RS), the ultimate andpentultimate enzymes of the spinach riboflavin biosynthetic pathway havebeen cloned by use of function complementation of E. coli auxotrophs.

Nucleic acid fragments that respectively encode LS protein and RSprotein have been isolated from plants and fungi. LS and RS genes fromother plants and fungi can now be identified by comparison of randomcDNA sequences to the sequences provided by Applicants. The inventionincludes assays using these nucleic acid fragments to screen for cropprotection chemicals related to the enzymatic pathway and methods foraltering the levels of production of LS and RS enzymes in a host cell.

In this disclose, a number of terms and abbreviations are used. Thefollowing definitions are provided.

"Lumazine synthase" is abbreviated as LS.

"Riboflavin synthase" is abbreviated as RS.

"Flavin mononucleotide" is abbreviated as FMN.

"Flavin adenine dinucleotide" is abbreviated as FAD.

"Polymerase chain reaction" is abbreviated PCR.

"Expressed sequence tag" is abbreviated EST.

"Dimethyl sulfoxide" is abbreviated DMSO.

"6,7-Dimethyl-8-(1'-D-ribityl)lumazine" is abbreviated DMRL.

"4-Ribitylamino-5-amino-2,6-dihydroxypyrimidine" is abbreviated RAADP.

"3,4-Dihydroxybutanone 4-phosphate" is abbreviated DHBP.

"Isopropyl-1-thio-β-D-galactopyranoside" is abbreviated IPTG.

"Sodium dodecylsulfate-polyacrylamide gel electrophoresis" isabbreviated SDS-PAGE.

"Open reading frame" is abbreviated ORF.

An "isolated nucleic acid fragment" is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA genomic DNA or synthetic DNA.

"Mature" protein refers to a functional LS or RS enzyme without itstransit peptide. "Precursor" protein refers to the mature protein with anative or foreign transit peptide. The term "transit peptide" refers tothe amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its uptake by organelles such as plastids orchloroplasts.

"Auxotrophy" refers to the nutritional requirements necessary forgrowth, sporulation and crystal production of the microorganism. For thepurpose of this invention, the term "auxotroph" is defined herein tomean an organism which requires the addition of riboflavin for growth.

The terms "host cell" and "host organism" refer to a cell capable ofreceiving foreign or heterologous genes and expressing those genes toproduce an active gene product. Suitable host cells includemicroorganisms such as bacteria and fungi, as well as plant cells.

The terms "lumazine synthase" or "LS" are used interchangeably with"6,7-dimethyl-8-ribityllumazine synthase" and refer to a plant or fungalenzyme that catalyzes the condensation of 3,4-dihydroxy-2-butanone4-phosphate with 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP)to yield orthophosphate and 6,7-dimethyl-8-(1'-D-ribityl)-lumazine(DMRL).

The terms "riboflavin synthase" or "RS" refer to a plant or fungalenzyme that catalyzes the dismutation of6,7-dimethyl-8-(1'-D-ribityl)-lumazine (DMRL) to yield riboflavin and3,4-dihydroxy-2-butanone 4-phosphate (DHBP) with4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP).

The term "metabolic activity" refers to the normal cellular activityneeded to support growth. As used herein agents such as crop protectionchemicals that will inhibit metabolic activity will also generallyinhibit cell growth.

The term, "substantially similar" refers to nucleic acid fragmentswherein changes in one or more nucleotide bases result in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. "Substantially similar" alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. "Substantially similar" also refers to modifications of thenucleic acid fragments of the instant invention such as deletion orinsertion of one or more nucleotide bases that do not substantiallyaffect the functional properties of the resulting transcript vis-a-visthe ability to mediate alteration of gene expression by antisense orco-Suppression technology or alteration of the functional properties ofthe resulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary sequences.

A "substantial portion" refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat potypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol.Biol. 215:403-410 (1993); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides (generally 12 bases orlonger) may be used as amplification primers in PCR in order to obtain aparticular nucleic acid fragment comprising the primers. Accordingly, a"substantial portion" of a nucleotide sequence comprises enough of thesequence to afford specific identification and/or isolation of a nucleicacid fragment comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular plant proteins. The skilled artisan, having the benefitof the sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for the purpose known to thoseskilled in the art. Accordingly, the instant invention comprises thecomplete sequences as reported in the accompanying Sequence Listing, aswell as substantial portions of those sequences as defmed above.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% identity with the geneto be suppressed. Moreover, alterations in a gene which result in theproduction of a chemically equivalent amino acid at a given site, but donot effect the functional properties of the encoded protein, are wellknown in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products. Moreover, the skilledartisan recognizes that substantially similar sequences encompassed bythis invention are also defined by their ability to hybridize, understringent conditions (0.1× SSC, 0.1% SDS, 65° C.), with the sequencesexemplified herein. Preferred substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are 80% identical to the DNA sequence of the nucleicacid fragments reported herein. More preferred nucleic acid fragmentsare 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are 95%identical to the DNA sequence of the nucleic acid fragments reportedherein.

"Codon degeneracy" refers to redundancy in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the LS or RS biosyntheticenzymes as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:38. The skilled artisan iswell aware of the "codon-bias" exhibited by a specific host cell inusage of nucleotide codons to specify a given amino acid. Therefore,when synthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

The term "percent identity" refers to the percent of identical matchesof either amino acids or bases when comparing either two amino acidsequences or two nucleotide sequences.

The term "complementary" is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Hencewith respect to DNA, adenosine is complementary to thymine and cytosineis complementary to guanine.

"Synthetic genes" can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. "Chemically synthesized", as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

"Gene" refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5' non-codingsequences) and following (3' non-coding sequences) the coding sequence."Native gene" refers to a gene as found in nature with its ownregulatory sequences. "Chimeric gene" refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. "Endogenous gene" refers to a native gene in its naturallocation in the genome of an organism. A "foreign" gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A "transgene" isa gene that has been introduced into the genome by a transformationprocedure.

"Coding sequence" refers to a DNA sequence that codes for a specificamino acid sequence. "Regulatory sequences" refer to nucleotidesequences located upstream (5' non-coding sequences), within, ordownstream (3' non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

"Promoter" refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3' to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an "enhancer" is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as "constitutive promoters". New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (Biochemistry of Plants 15:1-82 (1989)). It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The "translation leader sequence" refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed MRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to MRNA, MRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner et al., Mol. Biotech. 3:225(1995)).

The "3' non-coding sequences" refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3' end of the mRNA precursor. The use of different 3' non-codingsequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680(1989).

"RNA transcript" refers to the product resulting from RNApolymerasecatalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. "Messenger RNA" (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. "cDNA" refers to a double-stranded DNA that iscomplementary to and derived from mRNA. "Sense" RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. "Antisense RNA" refers to PI RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5' non-coding sequence, 3'non-coding sequence, introns, or the coding sequence. "FunctionalRNA"refers to antisense RNA, ribozyme RNA. or other RNA that is nottranslated yet has an effect on cellular processes.

The term "operably linked" refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term "expression", as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. "Antisense inhibition" refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. "Overexpression" refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. "Co-suppression"refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

"Altered levels" refers to the production of gene product(s) inorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

"Transformation" refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as "transgenic" organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al., Meth. Enzymol. 143:277 (1987)) andparticle-accelerated or "gene gun" transformation technology (Klein etal., Nature, London 327:70-73 (1987); U.S. 4,945,050).

"Chemical compound of interest" and "test compound" refer to thematerial which is being screened in the instant assay to assess itspotential as an herbicide or fungicide crop protection chemical.

A spinach LS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturespinach LS cDNA is provided in SEQ ID NO:1, and the deduced amino acidsequence is provided in SEQ ID NO:2. LS genes from other plants can nowbe identified by comparison of random cDNA sequences to the spinach LSsequence provided herein.

A tobacco LS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturetobacco LS cDNA is provided in SEQ ID NO:3, and the deduced amino acidsequence is provided in SEQ ID NO:4. LS genes from other plants can nowbe identified by comparison of random cDNA sequences to the tobacco LSsequence provided herein.

An arabidopsis LS has been isolated and identified by comparison ofrandom cDNA sequences to the GenBank database using the BLAST algorithmswell known to those skilled in the art. The nucleotide sequence ofmature arabidopsis LS cDNA is provided in SEQ ID NO:5, and the deducedamino acid sequence is provided in SEQ ID NO:6. LS genes from otherplants can now be identified by comparison of random cDNA sequences tothe arabidopsis LS sequence provided herein.

A Magnaporthe grisea LS has been isolated and identified by comparisonof random cDNA sequences to the GenBank database using the BLASTalgorithms well known to those skilled in the art. The nucleotidesequence of Magnaporthe grisea LS cDNA is provided in SEQ ID NO:37, andthe deduced amino acid sequence is provided in SEQ ID NO:38. LS genesfrom other fungi can now be identified by comparison of random cDNAsequences to the Magnaporthe grisea LS sequence provided herein.

A spinach RS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturespinach RS cDNA is provided in SEQ ID NO:7, and the deduced amino acidsequence is provided in SEQ ID NO:8. RS genes from other plants can nowbe identified by comparison of random cDNA sequences to the spinach RSsequence provided herein.

An arabidopsis RS has been isolated and identified by comparison ofrandom cDNA sequences to the GenBank database using the BLAST algorithmswell known to those skilled in the art. The nucleotide sequence ofmature arabidopsis RS cDNA is provided in SEQ ID NO:9, and the deducedamino acid sequence is provided in SEQ ID NO:10. RS genes from otherplants can now be identified by comparison of random cDNA sequences tothe arabidopsis RS sequence provided herein.

A Magnaporthe grisea RS has been isolated and identified by comparisonof random cDNA sequences to the GenBank database using the BLASTalgorithms well known to those skilled in the art. The nucleotidesequence of Magnaporthe grisea RS cDNA is provided in SEQ ID NO: 11, andthe deduced amino acid sequence is provided in SEQ ID NO:12. RS genesfrom other fungi can now be identified by comparison of random cDNAsequences to the Magnaporthe grisea RS sequence provided herein.

The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding a homologous LS and RS from the same orother plant or fungal species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction (PCR) or ligase chain reaction).

For example, LS or RS genes, either as cDNAs or genomic DNAs, could beisolated directly by using all or a portion of the instant nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired plant (or fungus) employing methodology well known to thoseskilled in the art. Specific oligonucleotide probes based upon theinstant LS or RS sequences can be designed and synthesized by methodsknown in the art (Maniatis supra). Moreover, the entire sequences can beused directly to synthesize DNA probes by methods known to the skilledartisan such as random primers, DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full-length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragment maybe used in PCR protocols to amplify longer nucleic acid fragmentsencoding homologous LS or RS genes from DNA or RNA. The polymerase chainreaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3' endof the mRNA precursor encoding plant LS or RS. Alternatively, the secondprimer sequence may be based upon sequences derived from the cloningvector. For example, the skilled artisan can follow the RACE protocol(Frohman et al., Proc. Natl. Acad. Sci., USA 85:8998 (1988)) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3' or 5' end. Primers oriented in the 3'and 5' directions can be designed from the instant sequences. Usingcommercially available 3' RACE or 5' RACE systems (BRL), specific 3' or5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci.,USA 86:5673 (1989); Loh et al., Science 243:217 (1989)). Productsgenerated by the 3' and 5' RACE procedures can be combined to generatefull-length cDNAs (Frohman et al., Techniques 1:165 (1989)).

Finally, availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acids sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner et al., Adv.Immunol. 36:1 (1984); Maniatis).

The nucleic acid fragments of the instant invention may also be used tocreate transgenic plants in which the instant LS or RS proteins arepresent at higher or lower levels than normal. Such manipulations wouldconceivably alter the intracellular levels of riboflavin, hence theessential cofactors FAD and FMN, producing novel phenotypes of potentialcommercial value. Alternatively, in some applications, it might bedesirable to express the instant LS or RS proteins in specific planttissues and/or cell types, or during developmental stages in which theywould normally not be encountered.

Overexpression of the instant LS or RS may be accomplished by firstconstructing a chimeric gene in which the LS or RS coding region isoperably linked to a promoter capable of directing expression of a genein the desired tissues at the desired stage of development. For reasonsof convenience, the chimeric gene may comprise promoter sequences andtranslation leader sequences derived from the same genes. 3' Non-codingsequences encoding transcription termination signals must also beprovided. The instant chimeric genes may also comprise one or moreintrons in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric genes can then beconstructed. The choice of a plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., EMBO J.4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86(1989)), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of MRNA expression, Western analysis of protein expression, orphenotypic analysis.

For some applications it may be useful to direct the LS or RS proteinsto different cellular compartments or to facilitate their secretion fromthe cell. It is thus envisioned that the chimeric genes described abovemay be further modified by the addition of appropriate intracellular orextracellular targeting sequences to their coding regions. These includechloroplast transit peptides (Keegstra et al., Cell 56:247-253 (1989),signal sequences that direct proteins to the endoplasmic reticulum(Chrispeels et al., Ann. Rev. Plant Phys. Plant Mol. 42:21-53 (1991),and nuclear localization signals (Raikhel et al., Plant Phys. 100:1627-1632 (1992). While the references cited give examples of each ofthese, the list is not exhaustive and more targeting signals of utilitymay be discovered in the future. As described below, it is demonstratedin the present invention that plant LS and RS are both synthesized asnuclear-encoded precursor proteins with chloroplast targeting sequencesat their N-termini. Thus, these proteins appear to be good candidatesfor targeting to other cellular compartments. Alternatively, by simplyremoving their chloroplast transit peptides, it should be possible toexpress LS or RS exclusively in the plant cytosol.

It may also be desirable to reduce or eliminate expression of the LS orRS genes in plants for some applications. In order to accomplish this,chimeric genes designed for antisense or co-suppression of LS or RS canbe constructed by linking the genes or gene fragments encoding parts ofthese enzymes to plant promter sequences. Thus, chimeric genes designedto express antisense RNA for all or part of LS or RS can be constructedby linking the LS or RS genes or gene fragments in reverse orientationto plant promoter sequences. The co-suppression or antisense chimericgene constructs could then be introduced into plants via well knowntransformation protocols to reduce or eliminate the endogenousexpression of LS or RS gene products.

The LS or RS protein produced in heterologous host cells, particularlyin the cells of microbial hosts, can be used to prepare antibodies tothe enzymes by methods well known to those skilled in the art. Theantibodies would be useful for detecting the instant LS or RS protein insitu in cells or in vitro in cell extracts. Preferred heterologous hostcells for production of the instant LS or RS protein are microbialhosts. Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of the instant LS orRS. These chimeric genes could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe instant LS or RS protein.

Microbial host cells suitable for the expression of the instant LS andRS enzymes include any cell capable of expression of the chimeric genesencoding these enzymes. Such cells will include both bacteria and fungiincluding, for example, the yeasts (e.g., Aspergillus, Saccharomyces,Pichia, Candida, and Hansenula), members of the genus Bacillus as wellas the enteric bacteria (e.g., Escherichia, Salmonella, and Shigella).Methods for the transformation of such hosts and the expression offoreign proteins are well known in the art and examples of suitableprotocols may be found in Manual of Methods for General Bacteriology(Gerhardt et al., eds., American Society for Microbiology, Washington,D.C. (1994) or in Brock, T. D., Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass. (1989)).

Vectors or cassettes useful for the transformation of suitable microbialhost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5' of the gene which harbors transcriptional initiation controlsand a region 3' of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the genes encoding the RS or LS enzymes in the desiredhost cell are numerous and familiar to those skilled in the art.Virtually any promoter capable of driving these genes is suitable forthe present invention including but not limited to CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TP1 (usefulfor expression in Sacharomyces); AOX1 (useful for expression in Pichia);and lac, trp, 1P_(L), 1P_(R), T7, tac, and trc (useful for expression inE. coli). Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary, however, it is most preferred if included.

The instant LS and RS proteins can be used as tools to facilitate thedesign and/or identification of specific chemical agents that mightprove useful as herbicides or fungicides. This could be achieved eitherthrough the rational design and synthesis of potent enzyme inhibitorsthat result from structural and/or mechanistic information that isderived from the purified instant plant proteins, or through random invitro screening of chemical libraries. LS and RS catalyze the last twosteps of riboflavin biosynthesis in plants and microorganism, and arerequired for the production of FAD and FMN, essential prosthetic groupsfor a number of important redox enzymes. Consequently, it is anticipatedthat significant in vivo inhibition of any of the LS or RS proteinsdescribed herein will severely cripple cellular metabolism and likelyresult in plant or fungal death.

All or a portion of the nucleic acid fragments of the instant inventionmay also be used as probes for genetically and physically mapping thegenes that they are a part of, and as markers for traits linked toexpression of the instant LS or RS. Such information may be useful inplant breeding in order to develop lines with desired phenotypes.

For example, the instant nucleic acid fragments may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blots(Maniatis) of restriction-digested plant genomic DNA may be probed withthe nucleic acid fragments of the instant invention. The resultingbanding patterns may then be subjected to genetic analyses usingcomputer programs such as MapMaker (Lander et at., Genomics 1:174-181(1987)) in order to construct a genetic map. In addition, the nucleicacid fragments of the instant invention may be used to probe Southernblots containing restriction endonuclease-treated genomic DNAs of a setof individuals representing parent and progeny of a defined geneticcross. Segregation of the DNA polymorphisms is noted and used tocalculate the position of the instant nucleic acid sequence in thegenetic map previously obtained using this population (Botstein et al.,Am. J Hum. Genet. 32:314-331 (1980)).

The production and use of plant gene-derived probes for use in geneticmapping is described by Bematzky and Tanksley (Plant Mol. Biol. Reporter4:37-41 (1986)). Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al., Nonmammalian Genomic Analysis: APractical Guide, pp. 319-346, Academic Press (1996), and referencescited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequence may be used in direct fluorescence in situhybridization (FISH) mapping. Although current methods of FISH mappingfavor use of large clones (several to several hundred kb), improvementsin sensitivity may allow performance of FISH mapping using shorterprobes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification, polymorphismof PCR-amplified fragments (CAPS), allele-specific ligation, nucleotideextension reactions, Radiation Hybrid Mapping and Happy Mapping. Forthese methods, the sequence of a nucleic acid fragment is used to designand produce primer pairs for use in the amplification reaction or inprimer extension reactions. The design of such primers is well known tothose skilled in the art. In methods employing PCR-based geneticmapping, it may be necessary to identify DNA sequence differencesbetween the parents of the mapping cross in the region corresponding tothe instant nucleic acid sequences. This, however, is generally notnecessary for mapping methods. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usage and conditions.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 1989; and by T. J. Silhavy,M. L. Bennan. and L. W. Enquist, Experiments with Gene Fusions. ColdSpring Harbor Laboratory Press, Cold Spring, N.Y. (1984) and by Ausubel,F. M. et al., Current Protocols in Molecular Biology, pub. by GreenePublishing Assoc. and Wiley-lnterscience (1987).

Manipulations of genetic sequences were accomplished using the suite ofprograms available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).

The meaning of abbreviations is as follows: "sec" means second(s), "min"means minute(s), "h" means hour(s), "d" means day(s), "μ1" meansmicroliter, "mL" means milliliters, "L" means liters, "mM" meansmillimolar, "M" means molar, "mmol" means millimole(s).

Example 1 PCR-Cloning of E. coli LS and RS

Gene specific PCR primers were used to amplify the E. coli LS and RSgenes from genomic DNA, while adding unique restriction sites to theirflanking regions for subsequent ligation into high copy number plasmids.The primers used for this purpose were based on the published DNAsequences of the E. coli LS and RS genes (GenBank accession numbersX64395 and X69109, respectively) and consisted of the followingnucleotides:

Primer 1-(SEQ ID NO:13):

5'-CGA AGG AAG Acc atg gCC ATT ATT GAA GCT AAC GTT GC-3'

Primer 2-(SEQ ID NO: 14):

5'-ATC TTA CTg tcg acT TCA GGC CTT GAT GGC TET C-3'

Primer 3-(SEQ ID NO:15):

5'-ACT CAT TTA cca tgg CTA CGG GGA TEG TAC AGG GC-3'

Primer 4-(SEQ ID NO:16):

5'-ATC TTA CTg tcg acT TCA GGC TTC TGT GCC TGG TT-3'

The underlined bases hybridize to the target genes, while lower caseletters indicate the restriction sites (NcoI or SalI) that were added tothe ends of the PCR primers.

Amplification of the LS gene was achieved using Primers 1 and 2, andgenomic DNA from E. coli strain W3110 (Campbell et al., Proc. Natl.Acad. Sci. 75:2276-2284 (1978)). Primer 1 hybridizes at the start of thegene and introduces a NcoI site at the protein's initiation codon, whilePrimer 2 hydridizes at the opposite end and provides a SalI site justpast the termination codon. The 100-μl PCR reactions contained ˜100 ngof genomic DNA and both primers at a final concentration of 0.5 μM. Theother reaction components were provided by the GeneAmp PCR Reagent Kit(Perkin Elmer), according to the manufacturer's protocol. Amplificationwas carried out in a DNA Thermocycler 480 (Perkin Elmer) for 28 cycles,each comprising 1 min at 94° C., 2 min at 53° C. and 2 min at 72° C.Following the last cycles there was a 7-min extension period at 72° C.The PCR product was cut with Ncol and SalI, and ligated into similarlydigested pGEM-5Zf (+) (Promega, Madison, Wis.). The latter was chosen asa suitable cloning vector since it lacks a NotI cleavage site afterdouble-digestion with NcoI and SalI (see below). The ligation reactionmixture was used to transform E. coli DH5α competant cells (GibcoBRL),and transformants were selected on LB media supplemented with 100 μg/mLampicillin.

The E. coli RS gene was amplified from genomic DNA in a similar mannerusing Primers 3 and 4. The former introduces a NcoI site at theprotein's initiation codon, while the latter provides a SalI site justafter the stop codon. Subsequent steps, including ligation of the PCRproduct into pGEM-5Zf (+) and transformation of DH5α with the resultingconstruct were exactly as described above.

Plasmids harboring the cloned E. coli LS and RS genes were identified byrestriction digestion analysis. Plasmid DNA was isolated from a numberof ampicillin-resistant colonies using the Wizard DNA PurificationSystem (Promega, Madison, Wis.) and subjected to cleavage with NcoI andSalI. The samples were analyzed by agarose gel electrophoresis, and arepresentative plasmid for each gene, yielding inserts of the correctsize, was sequenced completely to verify the absence of PCR errors.Apart from those nucleotides at the 5' and 3' ends that wereintentionally altered for cloning purposes, the amplified E. coli LS andRS gene sequences were identical to those reported in the literature.

Example 2 Insertional Inactivation of the E. coli LS and RS Genes

In order to create bacterial auxotrophs lacking the ability tosynthesize riboflavin, the cloned E. coli LS and RS genes were renderednonfunctional through insertional inactivation. Briefly, a unique NotIsite was introduced in the middle of the coding region of each of thetarget genes, and a DNA fragment that confers kanamycin resistance wasligated into the engineered sites. The latter was provided by thecommerically available Kan^(r) GenBlock cartridge (Pharmacia), that wasmodified through PCR to add NotI cleavage sites at both of its ends.This modification was accomplished using Primers 5 and 6 in a standardPCR reaction, the underlined portions hybridize to the Kan^(r) GenBlock,and lower case letters indicate the NotI cleavage sites.

Primer 5-(SEQ ID NO: 17):

5'-AAC TAG ATC Agc ggc cgc AGC CAC GTT GTG TCT CAA A-3'

Primer 6-(SEQ ID NO: 18):

5'-GAC AAA CAT Agc ggc cgc TGA GGT CTG CCT CGT GAA-3'

Following amplification, the modified Kan^(r) GenBlock was cleaved withNotI, and the resulting fragment was purified by agarose gelelectrophoresis.

PCR primers were also used to introduce a unique NotI cleavage site inthe middle of the two target genes. This was accomplished through anapplication of the "inverse PCR" technique that is fully described byOchman, et al. in PCR Protocols: A Guide to Methods and Applications,(Innis et al., eds.) pp. 219-227, Academic Press, San Diego, Calif.,(1990). The targets for inverse PCR are usually double-stranded circularDNA molecules. However, in contrast to other PCR applications, the twoprimers are oriented away from each other such that their 3' ends areextended in opposite directions around the entire circular template. Ifthe primers are designed to hybridize immediately adjacent to eachother, a linear DNA fragment is produced that includes the entire vectorsequence and has as its starting and stopping points the original primerbinding sites. The net result is analogous to linearizing a circularplasmid at a specified location. By attaching appropriate nucleotidesequences to the nonhybridizing 5' ends of both PCR primers, it istherefore possible to introduce a unique restriction site at any desiredlocation within a circular template.

Primers 7 and 8 (which hybridize to nt 2273-2290 and nt 2243-2261 of theDNA sequence in GenBank accession number X64395, respectively) weredesigned to introduce a NotI cleavage site in the middle of the E. coliLS gene; the nucleotides that hybridize to the target gene areunderlined, and NotI cleavage sites are indicated in lower case letters.

Primer 7-(SEQ ID NO: 19):

5'-AAC TAG ATC Agc ggc cgc GGT ACG GTT ATT CGT GGT-3'

Primer 8-LS (SEQ ID NO:20):

5'-GAC AAA CAT Agc ggc ctc GTC GTA TTT ACC GGT-3'

Primers 9 and 10 (which hybridize to nt 1217-1233 and nt 1190-1208 ofGenBank accession number X69109, respectively) were used to introduce aNotI cleavage site in the middle of the E. coli RS gene.

Primer 9-(SEQ ID NO:21):

5'-AAC TAG ATC Agc ggc cgc ACC ACT GCT GAA GTG GC-3'

Primer 10)-RS (SEQ ID NO:22):

5'-GAC AAA CAT Agc ggc cgc GAC CTG ACA TTA AGT GTC C-3'

The circular templates for inverse PCR were the pGEM-5Zf (+) constructscontaining the E. coli LS and RS genes. The 100-μl PCR reactionscontained 0.5 ng of plasmid DNA and each of the appropriate primers at afinal concentration of 0.5 μM. Amplification was carried out in a DNAThermocycler 480 (Perkin Elmer) for 30 cycles, each comprising 50 sec at94° C., I min at 55° C., and 3 min at 72° C. The PCR products werecleaved with NotI and the resulting fragments were purified by agarosegel electrophoresis; the excised bands were of the expected size. Next,the purified fragments were recircularized with T4 DNA ligase (Novagen)to regenerate functional plasmids, and aliquots of the ligation reactionmixtures were used to transform E. coli DH5α competant cells (GibcoBRL).Growth was selected for on LB media containing ampicillin (100 μg/mL),and plasmid DNA was isolated from a number of transformants forrestriction digestion analysis with NotI, Sall, and NcoI. For each ofthe target genes, a representative plasmid yielding the correct cleavagepatterns with these enzymes was selected for further manipulation.

To insert the kanamycin resistance gene, the two plasmid constructsdescribed above were cleaved with NotI and purified by agarose gelelectrophoresis. Each of the fragments was then individually incubatedwith a 4-fold molar excess of the modified Kan^(r) GenBlock cartridge,and subjected to a standard ligation reaction in the presence of T4 DNAligase (Novagen). Aliquots of the ligation reaction mixtures were usedto transform E. coli DH5α competant cells (GibcoBRL), and growth wasselected for on LB plates containing kanamycin (30 μg/mL) and ampicillin(100 μg/mL). Plasmids harboring the disrupted E. coli LS and RS geneswere identified by restriction digestion analysis. The plasmids werecleaved with NcoI and Sall, and were then subjected to agarose gelelectrophoresis to check for the presence of the inserted kanamycinresistance gene. Representative plasmids, yielding fragments of thecorrect size, were selected for further manipulation. DNA sequenceanalysis of these plasmids confirmed that the kanamycin resistance genehad been inserted at the correct location in both target genes.

Example 3 Generation of E. coli LS and RS Auxotrophs

The insertionally inactivated E. coli LS and RS genes were liberatedfrom the plasmid constructs described above using NcoI and Sall andpurified by agarose gel electrophoresis. Each of the fragments was thenindividually introduced into E. coli strain ATCC 47002 (fully describedin Balbas et al., Gene 136:211-213 (1993), and isogenic with JC7623(described by Bachmann, B., in E. coli and Salmonella typhimurium:Cellular and Molecular Biology (Niedhardt et al., eds.) p. 2466.American Society of Microbiology Washington. D.C. (1987)) byelectroporatation using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) according to the manufacturer's protocol.The choice of this strain as the initial recipient for gene replacementwas based on its well established hyper-rec phenotype and relatedability to undergo high frequency double-crossover homologousrecombination (Wyman et al., Proc. Nat. Acad. Sci. USA 82:2880-2884(1985); Balbas et al., Gene 136:211-213 (1993); Balbas et al., Gene172:65-69 (1996)). Thus, it was anticipated that the insertionallyinactivated E. coli LS and RS genes would efficiently replace theirfunctional chromosomal counterparts in ATCC 47002 under kanamycinselection.

Following electroporation, the transformed cells were resuspended in 1.0mL of S.O.C. media (GibcoBRL) that was supplemented with riboflavin (400μg/mL), and incubated for 1 h at 37° C. Kanamycin resistance was thenselected for on LB plates at 37° C. that contained both riboflavin (400μg/nL) and kanamycin (30 μg/mL); colonies appeared 24-48 h later.Phenotypic detection of the correct chromosomal integration event wasaccomplished through replica-plating experiments. Riboflavin auxotrophsresulting from double-crossover homologous recombination of either ofthe disrupted target genes would be expected to be resistant tokanamycin, sensitive to ampicillin, and to exhibit growth only in thepresence of added riboflavin. Representative bacterial coloniesexhibiting this phenotype were selected for further study.

While ATCC 47002 is an excellent strain for creating E. coli"knockouts", its multiple mutations in the recBCD loci render itincapable of propagating ColE1-type plasmids (Balbas et al., Gene172:65-69 (1996)). Consequently, the riboflavin auxotrophs describedabove are not suitable for screening plasmid cDNA libraries byfunctional complementation. In order to achieve this goal it wastherefore necessary to move the insertionally inactivated LS and RSgenes from the chromosome of ATCC 47002 to a suitable wild typebackground. This manipulation was accomplished through generalized phagetransduction using P1_(vir) and standard methodologies as fullydescribed by Miller, J. H., in Experiments in Molecular Genetics, pp.201-205, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1972). E. coli W3110 (Campbell et al., Proc. Natl. Acad. Sci.75:2276-2284 (1978)) was selected as the recipient strain for theinsertionally inactivated LS and RS genes. Following phage transduction,bacterial growth was selected for on LB media that was supplemented withkanamycin (35 μg/mL) and riboflavin (400 μg/mL). Stable transductantsharboring the disrupted E. coli LS or RS genes were then identifiedthrough replica-plating experiments analogous to those described abovefor ATCC 47002. Thus, individual colonies were patched onto platescontaining LB media, sodium citrate (7.5 mM), magnesium sulfate (1.5mM), and kanamyacin (35 μg/mL), with or without riboflavin (400 μg/mL).The LS and RS riboflavin auxotrophs that were selected for further studyand subsequent complementation cloning (see below) were only able togrow in the presence of added riboflavin, and were not resistant toampicillin (100 μg/mL) or streptomycin (25 μg/mL); sensitivity tostreptomycin is characteristic of W3110, but not of ATCC 47002.

Example 4 Cloning of Spinach LS and RS Genes Through FunctionalComplementation

A spinach (Spinacea oleracea) cDNA expression library in Lambda Zap IIwas obtained from Stratagene (La Jolla, Calif.), and subjected to massexcision according to the manufacturer's protocol. Upon excision, theliberated cDNA inserts are contained in the plasmid vector pBluscriptwhich confers resistance to amipicillin, and allows their expression inE. coli upon induction with isopropyl-1-thio-β-D-galactopyranoside(IPTG). The resulting mixture of excised plasmids was thenelectroporated into the E. coli LS and RS riboflavin auxotrophs (bothderivatives of W3110) using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) and the manufacturer's conditions. Thetransformed cells were selected for growth in the absence of addedriboflavin, on plates that contained B agar (LB media containing sodiumcitrate (7.5 mM), magnesium sulfate (1.5 mM), kanainyacin (35 μg/mL),ampicillin (100 μg/ML), and IPTG (0.6 mM)). Following a 48-hr incubationperiod at 37° C., bacterial growth was observed andriboflavin-independant colonies were recovered at frequencies of about4.2×10⁻⁶ and about 3.1×10⁻⁷, for the E. coli LS and RS auxotrophs,respectively. For each of the target genes, plasmid DNA was isolatedfrom a representative colony and subjected to further analysis; both ofthe selected plasmids were capable of transforming the appropiate E.coli auxotroph to riboflavin prototrophy at high frequency. The cDNAinserts contained in these plasmids were then sequenced completely on anABI 377 automated sequencer (Applied Biosystems), using fluorescentdideoxy terminators and custom-designed primers.

The approximately 1.2 kbp spinach cDNA insert that rescued the E. coliRS auxotroph clearly encodes a riboflavin synthase. The nucleotidesequence of the open reading frame (ORF) for this protein and itspredicted primary amino acid sequence are set forth in SEQ ID NO:33 andSEQ ID NO:34, respectively. While there are unmistakable similaritiesbetween the cloned plant protein and known microbial RS homologs,including those from E. coli, B. subtilis P. leiognathi, P. phosphoreumand S. cerevisiae (GenBank accession numbers, X69109, X51510, M90094,L11391 and Z2621, respectively), there are also some significantdifferences. The most obvious one is that the spinach RS is a muchlarger protein. In comparison to its counterparts in microorganisms, itpossesses an additional 69 amino acids at its N-terminus (FIG. 1). ThisN-termninal polypeptide extension is relatively basic, rich in Ser andThr residues, and as such, resembles a chloroplast transit peptide(Gavel et al., FEBS Lett. 261:455-458 (1990)). This observationsuggested that the spinach RS is synthesized as a nuclear-encodedprecursor protein, and is subsequently targeted to chloroplasts where itis proteolytically processed to its mature form. Indeed, based on thesequence alignments shown in FIG. 1 (generated with the GeneticsComputer Group Pile up program), the predicted cleavage-site formaturation occurs between amino acid residues 69 and 70 of the spinachRS precursor, giving rise to a mature polypeptide with a molecular massof 22.8 kDa (SEQ ID NO: 8). That this assignment is correct is stronglysupported by the fact that all known microbial RS homologs start withthe pentapeptide motif Met-Phe-Thr-Gly-Ile, which is apparently criticalfor function (Santos et al., J. Biol. Chem. 270:437-444 (1995)).

The notion that mature spinach RS is localized in chloroplasts is alsosupported by experimental evidence. Thus, antibodies directed againstthe purified recombinant protein (See below) specifically interact witha polypeptide of the expected size when spinach chloroplast extracts aresubjected to SDS-PAGE and Western analysis. Other experiments haveclearly demonstrated that the spinach RS precursor is imported intoplastids where it is proeolytically cleaved to its mature form. In thesestudies, the full-length spinach RS precursor was labeled with [S³⁵]-methionine through in vitro transcription/translation of the clonedgene, and subjected to in vitro protein import assays (Cline et al., J.Biol. Chem. 260, 3691-3696 (1988): Viitanen et al., J. Biol. Chem. 263,15000-15007 (1988)) using intact isolated spinach chloroplasts.

Of the various microbial homologs that are shown in FIG. 1, the maturespinach RS (SEQ ID NO: 8) shows the greatest similarity to the yeastprotein at the primary amino acid sequence level (e.g., approximately47% identity). In contrast, the mature spinach RS is approximately only35%, 42%, 34%, and 40% identical to the corresponding proteins from E.coli, B. subtilis, P. leiognathi, and P. phosphoreum, respectively.Taking into account the N-termninal chloroplast targeting sequence thatis unique to the plant protein the evolutionary divergence is evengreater.

Similar observations were made with the spinach cDNA insert that wascapable of restoring riboflavin prototrophy to the E. coli LS auxotroph.In this case, the approximately 1.3 kbp DNA fragment of the rescuingplasmid contained an ORF that encoded a much larger than normal LShomolog, the DNA sequence and predicted amino acid sequence of thelatter are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively.Analogous to the situation with the spinach RS precursor, the additionallength of the cloned spinach LS (relative to its homologs inmicroorganisms, including yeast) is entirely attributable to anN-terminal chloroplast transit peptide-like extension (FIG. 2). Thus, atleast the last two steps of higher plant riboflavin biosynthesis takeplace in chloroplasts. Although immunolocalization and chloroplastprotein import experiments (similar to those described above for spinachRS) have demonstrated that the spinach LS precursor is also targeted toplastids, it is more difficult in this case to predict with certaintythe exact start of the mature protein. Even amongst known microbial LShomologs it is apparent that the first 15-20 N terminal amino acidresidues are poorly conserved. Nevertheless, from the sequencealignments in FIG. 2, it is likely that the critical cleavage event formaturation occurs between Ala66 and Val67 of the spinach LS precursor,to yield a polypeptide with a predicted molecular mass of approximately16.5 kDa. While this notion remains to be determined experimentally, thepredicated amino acid sequence of the mature spinach LS based on thisassignment is given in SEQ ID NO:2. Note that even without itschloroplast targeting sequence, the mature spinach LS is only 49%, 47%,44%, 43% and 29% identical to its counterparts in E. coli, A.pleuropneumoniae, B. substilis, P. phosphoreum and S. cerevisiae,respectively (e.g., the other proteins shown in FIG. 2).

Example 5 Expression of Mature Spinach LS and RS in E. coli

The chloroplast targeting sequence of the cloned spinach RS precursor,identified in Example 4, was removed through a standard PCR reactionusing primers 11 (SEQ ID NO:23) and 12 (SEQ ID NO:24). Primer 11 (5'-CTACTC ATT TCA TAT GTT CAC TGG CAT TGT TGA A-3') (SEQ ID NO:23) hydridizesto nt 208-228 of the spinach RS precursor (SEQ ID NO:33). It wasdesigned to initiate protein synthesis in E. coli at the predicted startof the mature protein (SEQ ID NO:8), and incorporates a unique NdeI siteupstream from the initiator Met residue for cloning purposes. Primer 12(5'-CAT CTT ACT GGA TCC ACT ATG TGA ATT TGG TAG GAT C-3') (SEQ ID NO:24)hybridizes at the other end of the ORF to nt 820-840 and introduces aunique BamHI site just past the protein's stop codon. The target for PCRamplification was the purified plasmid containing the cDNA insert forthe spinach RS precursor. The predicted PCR product encodes thefull-length mature spinach RS without any modifications (SEQ ID NO:8).

A similar strategy was employed to remove the transit peptide from thecloned spinach LS precursor for expression in E. coli. However, in thiscase, it was also necessary to provide the truncated plant protein withan initiator Met residue at the predicted transit peptide cleavage-sitesince a naturally occurring one was lacking. This was accomplished usingprimers 13 (SEQ ID NO:25) and 14 (SEQ ID NO:26) in a standard PCRreaction with purified plasmid containing the cDNA insert for thespinach LS precursor. Primer 13 (5'-CTA CTC ATT TCA TAT GAA CGA GCT TGAAGG TTA TGT CAC-3') (SEQ ID NO:25) hybridizes to nt 205-224 of thespinach LS precursor (SEQ ID NO:27), and introduces an initiator Metresidue at the position currently occupied by Val67 (SEQ ID NO:28), thepredicted start of the mature protein (SEQ ID NO:2). This primer alsoprovides a unique NdeI site at the introduced initiator Met residue andchanges the second amino acid from Arg to Asn. It was reasoned thatthese changes would not compromise enzyme activity, and might actuallyimprove bacterial expression of the modified plant protein, since boththe E. coli and B. subtilis LS homologs start with the dipeptidesequence, Met-Asn. Primer 14 (5'-CAT CTT ACT GGA TCC ATC AGG CCT TCA AATGAT GTT CG-3') (SEQ ID NO:26) hybridizes at the other end of the spinachLS precursor to nt 648-667, and provides a unique BamHI site just pastthe termination codon. Thus, with the exception of the first two aminoacids, the PCR fragment generated with primers 13 and 14 will encode apolypeptide with the same primary amino acid sequence as that shown inSEQ ID NO:2.

Following amplification of the two target genes, the PCR fragments werecleaved with NdeI and BamHI, and were individually ligated intosimilarly digested pET-24a (+) (Novagen). The latter is a high-level E.coil expression vector that is under the control of the T7 promoter.Aliquots of the ligation reaction mixtures were then used to transformE. coli BL21 (DE3) using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) according to the manufacturer's protocol.The transformed cells were plated on LB media containing kanamyacin (50μg/mL) and incubated at 37° C. to obtain single colonies. Clonesharboring plasmids with the correct inserts were identified through PCRreactions using individual resuspended colonies and the appropriateprimer pairs (i.e., primers 11 and 12 for the mature spinach RSconstruct and primers 13 and 14 for the mature spinach LS construct).Following this procedure, a representative clone for each of the targetgenes was selected for further manipulation and these two strains wereused for the production of recombinant proteins as described below.Plasmid DNA from these strains was sequenced completely to check for PCRerrors, and in both cases, none were found.

For overexpression of the mature spinach LS and RS proteins, theBL21(DE3) strains described above were grown in LB media containingkanamycin (50 μg/mL) at 37° C. The cells were induced with IPTG (1 mM)at an A₆₀₀ nm of about 1.0, and were harvested 3 h later bycentrifugation. Both plant proteins were well expressed in the bacterialhost at levels exceeding 15% of the total soluble protein. Subsequentmanipulations were at 0-4° C. Cell pellets containing recombinantspinach RS were resuspended in 2.5 vol of 0.1 M potassium phosphate (pH7.2), 10 mM sodium sulfite, 10 mM EDTA, and passed twice through aFrench pressure cell at 20,000 psi. Debris was removed by centrifugation(10⁵ ×g, 1 h), and the cell-free extract, containing 44 mg ofprotein/mL, was supplemented with glycerol (5%) and stored at -80° C.for subsequent use. Protein concentrations were determined by the methodof Lowry et al. (Lowry et al., J. Biol Chem. 193:265-275 (1951)), usingBSA as a standard. Cell pellets containing recombinant spinach LS weredisrupted in an identical manner, but the buffer used for cellresuspension was 100 mM Tris-HCl (pH 7.7), 5 mM MgSO₄, 0.03 mg/mL DNAseI (Sigma), 0.5 mM phenylmethylsulfonyl flouride, 1 mM dithiothreitol andthe protein concentration of the cell-free extract was 39 mg/mL.SDS-PAGE analysis of the cell-free extracts revealed that both plantproteins were well expressed in the bacterial host, at levels exceeding20% of the total soluble protein.

Example 6 Purification of Recombinant Mature Spinach RS

An aliquot (0.5 mL) of E. coli cell-free extract containing therecombinant spinach RS was rapidly thawed to room temperature, diluted1:1 with deionized water, and filtered through a 0.2 μm Acrodisc filter(Gelman Sciences, Cat. No. 4192). The entire sample was then applied toa Mono Q HR 5/5 column (Pharmacia Biotech Inc), preequilibrated at 25°C. with Buffer Q (50 mM Tris-HCI, pH 7.7, 10 mM sodium sulfite, 1 mMEDTA). The column was developed at 1.0 mL/min with a linear gradient (30mL) of 0-1.0 M NaCl (in Buffer Q), and 1-mL fractions were collected.The position in the gradient where spinach RS elutes was determined bySDS-PAGE (Laemmli U., Nature 227:680-685 (1970)) using 15% gels andCoomassie Blue staining. Subsequently, column fractions eluting between0.167-0.233 M NaCl were pooled and concentrated in a Centricon-10(Amicon Inc.) at 4° C. to a final volume of 450 μL. In the next step,half of this material was applied to a 7.5×600 mm TSK G3000SW gelfiltration column (TOSOH Corp.) that was preequilibrated with Buffer Qcontaining 0.3 M NaCl. The column was developed at a flow rate of 1.0mL/min (25° C.), and highly purified spinach RS eluted between 15.2-16.2min. The latter was kept on ice while the remaining half of the samplewas processed in an identical manner. The peak fractions from the twogel filtration columns were pooled, supplemented with glycerol (5%),concentrated to 6.6 mg of protein/mL, and stored at -80° C. forsubsequent use. The yield of purified protein was 2.9 mg, correspondingto 13% of the total protein present in the cell-free extract. Visualinspection of overloaded Coomassie-stained gels suggested the finalpreparation was >95% pure.

Edman degradation of the purified recombinant spinach RS revealed thatits first 21 amino acids are identical to those of the protein shown inSEQ ID NO:8, in accord with the PCR strategy that was employed in itsconstruction. This further indicates that the recombinant protein'sN-terminus remained intact during the purification procedure. Asdetermined by electrospray ionization mass spectrometry, the protomermolecular mass of the purified recombinant spinach RS was 22808.3daltons, a value that is in excellent agreement with that predicted fromits DNA sequence (22807.26 daltons). Similar to the E. coli (Bacher etal., J. Biol Chem. 255:632-637 (1980)) and yeast (Santos et al., J.Biol. Chem. 270:437-444 (1995)) RS homologs, both of which are trimersin the native state, the recombinant spinach RS eluted during analyticalgel filtration with an apparent molecular mass of 65 kDa. Moreimportant, the mature plant protein is catalytically active. In the invitro enzyme assay described below, the purified recombinant spinach RSexhibited a turnover number of approximately 0.08/sec at 25° C. (basedon protomer). By way of comparison, the reported turnover numbers for S.cerevisiae (Santos et al., J. Biol. Chem. 270:437-444 (1995)) and B.subtilis (Bacher et al., J. Biol. Chem. 255:632-637 (1980)) RS, at 37°C., are 0.13/sec and 0.33/sec, respectively. Assuming that the enzymereaction is characterized by a Q10 (temperature coefficient) of at least2, these observations suggest that the purified recombinant spinach RSis probably fully active.

Example 7 Purification and Physical Properties of Recombinant MatureSpinach LS

An aliquot (0.5 mL) of E. coli cell-free extract containing therecombinant spinach LS was rapidly thawed to room temperature, diluted1:1 with deionized water, and filtered through a 0.2 μm Acrodisc filter(Gelman Sciences, Cat. No. 4192). The entire sample was thenfractionated on a Mono Q HR 5/5 column, using the same buffers andconditions that were described above for recombinant spinach RS. Thematerial eluting between 0.367-0.433 M NaCl was pooled, concentrated to450 μL. and subjected to gel filtration chromatography exactly asdescribed above for the recombinant spinach RS. Highly purified spinachLS emerged from the sizing column as a sharp peak eluting between10.15-10.85 min, and this material was supplemented with glycerol (5%),concentrated to 12.1 mg of protein/mL, and stored at -80° C. forsubsequent use. The final yield of purified protein was 4.3 mg (nearly22% of the total protein present in the cell-free extract) and thepreparation was essentially homogeneous as judged from Coomassie-stainedgels.

The nucleotide sequence of the mature recombinant spinach LS predicts apolypeptide of 16534.71 daltons, a value that is virtually identical tothat which was obtained with the purified protein using electrosprayionization mass spectrometry (16536.3 daltons). Assuming that plant LSforms a hollow, spherical particle, comprised of 60 identical subunits,like the E. coli (Mortl et al., J. Biol. Chem. 271:33201-33207 (1996))and B. subtilis (Bacher et al., J. Biol Chem. 255:632-637 (1980))homologs, its native molecular mass should be about 992 kDa. Indeed,during analytical gel filtration, the purified recombinant spinach LSexhibited an apparent molecular mass of about 823 kDa. It would thusappear that the quaternary structure of this riboflavin biosyntheticenzyme has been highly conserved in the evolution from bacteria tohigher plants. Edman degradation confirmed that the first two N-terminalamino acid residues of the recombinant protein had been correctlyaltered through PCR from Val-Arg to Met-Asn as previously described.Despite these substitutions and removal of the chloroplast targetingsequence, the purified recombinant spinach LS still retained catalyticactivity. At 25° C., using the in vitro enzyme assay described below,its turnover number was 0.013/sec (based on protomer). This value is inreasonable agreement with the turnover numbers reported for the purifiedE. coli, yeast, and B. subtilis enzymes (0.06/sec), which were allmeasured at 37° C. (Kis et al., Biochemistry 34:2883-2892 (1995); Mortlet al., J. Biol. Chem. 271:33201-33207 (1996)). Thus, it is very likelythat the recombinant spinach LS is also fully active.

Example 8 Preparation of Substrates For RS and LS

6,7-Dimethyl-8-(1'-D-ribityl)lumazine (DMRL) was synthesized aspreviously described (Plaut, G. W. E. and Harvey, R. A., Methods inEnzymology (McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp.515-538, Academic Press, NY (1971)) and purified by HPLC on a C-18column developed with a water to methanol gradient. The purifliedmaterial was taken to dryness in a rotovap and stored at -20° C. forsubsequent use.

4-Ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) was prepared from4-ribitylamino-5-nitroso-2,6-dihydroxypyrimidine by catalytichydrogenation (Plaut. G. W. E. and Harvey, R. A., Methods in Enzymoloyy(McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp. 515-538.Academic Press, NY (1971)). The 40-mL reaction mixture contained 0.4mmol of the latter compound, dissolved in 10 mM acetic acid, and 20 mgof 10% palladium on carbon. Following an overnight incubation period at25° C. (with gentle shaking, under 50 psi H₂) the catalyst was removedby filtration and the filtrate containing RAADP was stored in aliquotsat -80° C.

3,4-Dihydroxybutanone 4-phosphate (DHBP) was prepared enzymatically fromD-ribose 5-phosphate. The reaction mixture contained 50 mM Tris-HCl (pH7.5), 20 mM MgCl₂, 20 mM D-ribose 5-phosphate, 10 units/mLphosphoribo-isomerase (Sigma, Cat. No. P7434) and 0.3 units/mL E. coliDHBP synthase. After 2 h at 25° C., the reaction reached completion andaliquots of the solution containing DHBP were stored at -80° C.

Example 9 Riboflavin Synthase Assays

Riboflavin synthase assays were run using 1-mL reaction mixturescontaining 0.1 mM DMRL in 50 mM Tris-Cl (pH 7.5) at 25° C. Reactionswere initiated by the addition of purified recombinant spinach RS andinitial rates of the reactions were measured continuously at 470 nm. Amolar extinction coefficient (ε) of 9500 at 470 nm was used to calculatethe formation of riboflavin (Plaut, G. W. E. and Harvey, R. A., Methodsin Enzymology (McCormick, D. B. and Wright L. D., eds.) vol. 18, part B,pp. 515-538, Academic Press, NY (1971)).

Inhibitor screens were carried out in 96-well plates. Potentialinhibitory compounds were dissolved in dimethyl sulfoxide (DMSO) (10mg/mL) and then serially diluted with water to concentrations of 0.1mg/mL in 1% aqueous DMSO. Reaction mixtures (0.21 mL total) contained0.115 mL DMRL (0.035 mM) in 100 mM Tris-Cl (pH 7.5) and 0.085 mLpotential inhibitory compound (0.1 mg/mL) at 25° C. Before initiatingreactions with enzyme, the absorbance at 470 nm was recorded. Reactionswere initiated with 0.01 mL of purified recombinant spinach RS (0.28mg/mL) and after a 3 min incubation at 25° C. the plates were read at470 nm. The first absorbance reading is subtracted from the second toafford rates of riboflavin formation. Column 1 of the 96-well platescontained no compounds and the reactions in these wells served asuninhibited controls. Compounds that reduced the rate of riboflavinformation (in comparison to the uninhibited control reactions) werefollowed up with 1-mL confirmation assays where IC50's were determined.

Example 10 Lumazine Synthase Assays

Lumazine synthase assays were run using 1-mL reaction mixtures whichcontain 0.05 mM RAADP and 0.05 mM DHBP in 50 mM Tris-HCl (pH 7.5) at 25°C. Reactions were then initiated by the addition of purified recombinantspinach LS and initial rates of the reactions were monitoredcontinuously at an absorbance of 408 nm. A molar extinction coefficient(ε) of 10,000 at 408 nm was used to calculate the rate of formation ofDMRL (Plaut, G. W. E. and Harvey, R. A., Methods in Enzymology(McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp. 515-538,Academic Press, N.Y. (1971)).

Inhibitor screens are carried out in 96-well plates. Potentialinhibitory compounds are dissolved in 10% aqueous DMSO to aconcentration of 1 mg/mL. Reaction mixtures (0.21 mL total) are preparedby adding 0.005 mL potential inhibitory compound (1.0 mg/mL) to 0.195 mLof a solution containing 0.05 mM DHBP, 0.05 mM RAADP in 50 mM Tris-Cl(pH 7.5) at 25° C. Reactions are then initiated with 0.01 mL of purifiedrecombinant spinach LS (1.6 mg/mL) and progress of the reactions ismonitored continuously at 408 nm for 5 min. Column 1 of the 96-wellplates contains no test compounds and the rates of DMRL formation inthese wells serve as uninhibited controls. Compounds that reduce therates of DMRL formation (in comparison to the uninhibited controls) arefollowed up with 1-mL confirmation assays where IC50's are determined.

Example 11 Other Plant and Fungal RS and LS Genes

Using the methodologies described in Example 4, several other plant andfungal LS and RS cDNAs have been cloned through functionalcomplementation of the E. coli riboflavin auxotrophs. These include thenuclear-encoded precursor proteins for arabidopsis RS, tobacco LS, andarabidopsis LS and full-length mature RS and LS proteins from the riceblast fungus Magnaporthe grisea. The nucleotide sequences of the ORFsfor these proteins are respectively documented in SEQ ID NO:35, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:11 and SEQ ID NO:37, and theircorresponding amino acid sequences are given in SEQ ID NO:36, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO: 12, and SEQ ID NO:38, respectively. TheLamba cDNA expression libraries from which the arabidopsis LS and RSgenes and tobacco LS gene were cloned are commercially available fromStratagene (Cat. Nos. 937010 and 936002, respectively). The cDNAexpression library from which the Magnaporthe grisea RS gene wasobtained was contained in the Lambda ZipLox vector (GibcoBRL) betweenthe NotI and SalI cleavage sites of the polylinker region, and wasprepared from isolated mRNA using conventional methodologies (Maniatis).The cDNA expression library from which the Magnaporthe grisea LS genewas obtained was cloned between the EcoRI and XhoI sites of the plasmidvector pBluescript II SK (+), available from Stratagene, and was alsoprepared from isolated mRNA using standard procedures (Maniatis). Asdescribed above for cloning the spinach LS and RS precursor genes (as inExample 4), the various Lamba cDNA expression libraries were firstsubjected to mass excision to yield plasmid cDNA libraries, which werethen introduced into the E. coli LS and RS auxotrophs (W3110derivatives) via electroporation. Following this procedure,transformants were selected for growth in the absence of addedriboflavin (on plates containing B agar), and the cDNA inserts of therescuing plasmids were isolated and sequenced completely usingcustom-designed primers.

As shown in FIG. 3, the cloned spinach and arabidopsis RS are verysimilar. Both proteins are synthesized as larger molecular weightprecursors with a chloroplast targeting sequence at their N-terminus(boxed residues in FIG. 3). Although the transit peptides of the twoplant species are of comparable length, they are highly divergent andbear little resemblance to each other at the primary amino sequencelevel. In contrast, the mature spinach and arabidopsis RS are wellconserved, with nearly 70% of their residues being identical. Theseobservations are not surprising since most nuclear-encoded chloroplastproteins, including the precursor for the small subunit ofribulose-1,5-bisphosphate carboxylaseoxygenase (Mazur et al., Nucl.Acids Res. 13:2373-2386 (1985)), exhibit much greater species-to-speciesvariation in their chloroplast targeting sequence than in the matureportion of the molecule. The two plant RS homologs also possess a numberof polypeptide motifs that are not present in the bacterial and fungalRS homologs that have currently been sequenced. It is possible that someof these highly conserved regions that are unique to plants mightspecifically influence the catalytic and/or regulatory properties of thehigher plant RS, thereby providing valuable insight in the design ofenzyme inhibitors that could be useful as herbicides.

A comparison of the spinach, tobacco, and arabidopsis LS precursorproteins, also cloned by functional complementation, provides a similarpicture (FIG. 4). Again, the chloroplast transit peptides of threeprecursors are poorly conserved (boxed residues), while the matureproteins exhibit 72-76% identity at the amino acid sequence level.Additionally, all three plant proteins possess a unique stretch of aminoacid residues at their C-terminus (e.g., ASLFEHHLK [SEQ ID NO:39]), aregion of the polypeptide that is highly divergent in microbial LShomologs. That these nine residues are identical for three differentplant species, suggests that they might be of unique importance to thefunctionality of the higher plant proteins. Based on this observation,it is anticipated that further structural and mechanistic studies withthe purified plant LS proteins described herein will greatly assist therational design of enzyme inhibitors that are specifically herbicidal.

Until the present invention, the only fungal LS and RS homologs thathave been sequenced are those of S. cervasiae (Garcia-Ramirez et al., J.Biol. Chem. 270:23801-23807 (1995)); Santos et al., J. Biol. Chem.270:437-444 (1995)). These proteins bear little resemblence to theMagnaporthe grisea LS and RS proteins that were cloned in the presentwork by functional complementation. The two fungal RS homologs are only47% identical at the primary amino acid sequence level. While a similardegree of conservation is observed between the Magnaporthe grisea and S.cerevesiae LS proteins (51% identity), the former is significantlylonger than other known microbial homologs. Moreover, the additionallength of this protein is not due to the presence of a cleavableN-terminal targeting sequence as described above for the higher plant LSand RS precursors. Instead, it possesses a unique polypeptide segmentthat appears to have been inserted right about in the middle of theprotein; based on sequence alignments with other LS homologs, theadditional residues (consisting largely of Ser and Thr) correspond toamino acids 74-104 of the Magnaporthe grisea LS (SEQ ID NO:38). Thesignificance of this observation is not yet understood, but it couldhave practical application in the development of novel fungicides foruse in the treatment of rice blast.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  39                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  471 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature spinach LS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - GTTAGGGAGC TTGAAGGTTA TGTCACTAAA GCCCAGAGTT TCAGATTTGC CA -             #TTGTTGTG     60                                                                 - - GCTAGGTTCA ACGAATTTGT GACAAGACGA CTAATGGAAG GAGCTCTTGA CA -            #CTTTTAAG    120                                                                 - - AAATACTCTG TCAATGAAGA TATTGATGTT GTTTGGGTTC CTGGTGCTTA TG -            #AGCTAGGT    180                                                                 - - GTTACTGCAC AAGCACTTGG GAAATCAGGA AAATATCATG CTATTGTTTG TC -            #TTGGAGCT    240                                                                 - - GTGGTAAAAG GTGATACTTC ACACTATGAT GCTGTCGTTA ATTCTGCTTC CT -            #CTGGAGTA    300                                                                 - - CTGTCAGCTG GATTAAATTC AGGAGTACCT TGTGTCTTTG GTGTCCTTAC CT -            #GTGATAAC    360                                                                 - - ATGGATCAGG CCATAAATCG AGCTGGCGGG AAAGCGGGTA ATAAAGGAGC CG -            #AGTCAGCG    420                                                                 - - CTAACAGCTA TTGAAATGGC TTCGCTTTTC GAACATCATT TGAAGGCCTA A - #                471                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  156 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  unkn - #own                                                (D) TOPOLOGY:  unknown                                               - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature spinach LS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Val Arg Glu Leu Glu Gly Tyr Val Thr Lys Al - #a Gln Ser Phe Arg Phe       1               5  - #                 10 - #                 15              - - Ala Ile Val Val Ala Arg Phe Asn Glu Phe Va - #l Thr Arg Arg Leu Met                   20     - #             25     - #             30                  - - Glu Gly Ala Leu Asp Thr Phe Lys Lys Tyr Se - #r Val Asn Glu Asp Ile               35         - #         40         - #         45                      - - Asp Val Val Trp Val Pro Gly Ala Tyr Glu Le - #u Gly Val Thr Ala Gln           50             - #     55             - #     60                          - - Ala Leu Gly Lys Ser Gly Lys Tyr His Ala Il - #e Val Cys Leu Gly Ala       65                 - # 70                 - # 75                 - # 80       - - Val Val Lys Gly Asp Thr Ser His Tyr Asp Al - #a Val Val Asn Ser Ala                       85 - #                 90 - #                 95              - - Ser Ser Gly Val Leu Ser Ala Gly Leu Asn Se - #r Gly Val Pro Cys Val                  100      - #           105      - #           110                  - - Phe Gly Val Leu Thr Cys Asp Asn Met Asp Gl - #n Ala Ile Asn Arg Ala              115          - #       120          - #       125                      - - Gly Gly Lys Ala Gly Asn Lys Gly Ala Glu Se - #r Ala Leu Thr Ala Ile          130              - #   135              - #   140                          - - Glu Met Ala Ser Leu Phe Glu His His Leu Ly - #s Ala                      145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  474 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature tobacco LS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - GTTCGTCAGT TGACTGGTTC TGTTACCTCT GCCAAAGGCC ATCGCTTTGC TG -             #TTGTGGTT     60                                                                 - - GCACGTTTCA ATGATCTTAT CACCAAGAAG CTTTTGGAGG GAGCTTTGGA CA -            #CTTTCAAA    120                                                                 - - AATTACTCTG TTAGAGAGGA AGATATTGAT GTCGTGTGGG TTCCTGGCTG TT -            #TTGAAATC    180                                                                 - - GGTGTGGTTG CGCAACAGCT TGGAAAGTCG CAGAAATATC AAGCAATACT CT -            #GTATTGGG    240                                                                 - - GCTGTGATTA GAGGTGATAC GTCTCACTAT GATGCCGTCG TTAATGCTGC CA -            #CATCCGGA    300                                                                 - - GTACTTTCAG CAGGTCTAAA TTCTGGTACT CCTTGCATAT TTGGTGTTTT GA -            #CATGTGAT    360                                                                 - - ACCTTGGAGC AGGCTTTCAA TCGTGTCGGT GGGAAGGCTG GGAATAAAGG TG -            #CCGAAACA    420                                                                 - - GCGTTGACAG CTATTGAGAT GGCGTCTTTG TTTGAACACC ACTTAAAGGC TT - #AA              474                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  157 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature tobacco LS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - Val Arg Gln Leu Thr Gly Ser Val Thr Ser Al - #a Lys Gly His Arg Phe       1               5  - #                 10 - #                 15              - - Ala Val Val Val Ala Arg Phe Asn Asp Leu Il - #e Thr Lys Lys Leu Leu                   20     - #             25     - #             30                  - - Glu Gly Ala Leu Asp Thr Phe Lys Asn Tyr Se - #r Val Arg Glu Glu Asp               35         - #         40         - #         45                      - - Ile Asp Val Val Trp Val Pro Gly Cys Phe Gl - #u Ile Gly Val Val Ala           50             - #     55             - #     60                          - - Gln Gln Leu Gly Lys Ser Gln Lys Tyr Gln Al - #a Ile Leu Cys Ile Gly       65                 - # 70                 - # 75                 - # 80       - - Ala Val Ile Arg Gly Asp Thr Ser His Tyr As - #p Ala Val Val Asn Ala                       85 - #                 90 - #                 95              - - Ala Thr Ser Gly Val Leu Ser Ala Gly Leu As - #n Ser Gly Thr Pro Cys                  100      - #           105      - #           110                  - - Ile Phe Gly Val Leu Thr Cys Asp Thr Leu Gl - #u Gln Ala Phe Asn Arg              115          - #       120          - #       125                      - - Val Gly Gly Lys Ala Gly Asn Lys Gly Ala Gl - #u Thr Ala Leu Thr Ala          130              - #   135              - #   140                          - - Ile Glu Met Ala Ser Leu Phe Glu His His Le - #u Lys Ala                  145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  471 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature arabidopsis LS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - GTTCGCCATG TTACGGGGTC TCTTATCAGA GGCGAAGGTC TTAGATTCGC CA -             #TCGTGGTA     60                                                                 - - GCTCGTTTCA ATGAGGTTGT GACTAAGTTG CTTTTGGAAG GAGCGATTGA GA -            #CTTTCAAG    120                                                                 - - AAGTATTCAG TCAGAGAAGA AGACATTGAA GTTATTTGGG TTCCTGGCAG CT -            #TTGAAATT    180                                                                 - - GGTGTTGTTG CACAAAATCT TGGGAAATCG GGAAAATTTC ATGCTGTTTT AT -            #GTATCGGC    240                                                                 - - GCTGTGATAA GAGGAGATAC CACACATTAT GATGCTGTTG CCAACTCTGC TG -            #CGTCTGGA    300                                                                 - - GTACTTTCTG CTAGCATAAA TTCAGGCGTT CCATGCATAT TTGGTGTACT GA -            #CTTGCGAG    360                                                                 - - GACATGGATC AGGCTCTGAA TCGATCTGGT GGCAAAGCCG GCAATAAGGG AG -            #CTGAAACT    420                                                                 - - GCTTTGACGG CGCTCGAAAT GGCGTCGTTG TTTGAGCACC ACCTGAAATA G - #                471                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  156 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  unkn - #own                                                (D) TOPOLOGY:  unknown                                               - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # mature arabidopsis LS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - Val Arg His Val Thr Gly Ser Leu Ile Arg Gl - #y Glu Gly Leu Arg Phe       1               5  - #                 10 - #                 15              - - Ala Ile Val Val Ala Arg Phe Asn Glu Val Va - #l Thr Lys Leu Leu Leu                   20     - #             25     - #             30                  - - Glu Gly Ala Ile Glu Thr Phe Lys Lys Tyr Se - #r Val Arg Glu Glu Asp               35         - #         40         - #         45                      - - Ile Glu Val Ile Trp Val Pro Gly Ser Phe Gl - #u Ile Gly Val Val Ala           50             - #     55             - #     60                          - - Gln Asn Leu Gly Lys Ser Gly Lys Phe His Al - #a Val Leu Cys Ile Gly       65                 - # 70                 - # 75                 - # 80       - - Ala Val Ile Arg Gly Asp Thr Thr His Tyr As - #p Ala Val Ala Asn Ser                       85 - #                 90 - #                 95              - - Ala Ala Ser Gly Val Leu Ser Ala Ser Ile As - #n Ser Gly Val Pro Cys                  100      - #           105      - #           110                  - - Ile Phe Gly Val Leu Thr Cys Glu Asp Met As - #p Gln Ala Leu Asn Arg              115          - #       120          - #       125                      - - Ser Gly Gly Lys Ala Gly Asn Lys Gly Ala Gl - #u Thr Ala Leu Thr Ala          130              - #   135              - #   140                          - - Leu Glu Met Ala Ser Leu Phe Glu His His Le - #u Lys                      145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  633 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Mature Spinach RS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - ATGTTCACTG GCATTGTTGA AGAGATTGGC CGAGTTAAGC AAATGGGTTA TG -             #GCGAAGAC     60                                                                 - - GGTGGATTTC AGCTTAAAGT TGTAGGAGAC ATTGTCCTAA AAGATGTCAA TC -            #TTGGTGAC    120                                                                 - - AGTATCGCAG TTAATGGTAC ATGTCTAACT GTGACGGAAT TTGACACTAA AG -            #CGTCCGAA    180                                                                 - - TTTACTCTTG GGATAGCGCC TGAGACGCTT AGGAAGACGG CATTGATGGA TC -            #TCGAACCA    240                                                                 - - GGGTCAGTTG TTAATTTAGA AAGAGCCCTT TTGCCTTCTA CACGGATGGG TG -            #GTCACTTT    300                                                                 - - GTCCAGGGAC ATGTTGATGG GACAGGAGAA ATTGTATCAC TAGTTGAAGA AG -            #GTGATTCT    360                                                                 - - TTGTGGGTCA AGATAAAAAC AAGCCCAGAA ATACTGAGAT ACATTGTACC AA -            #AAGGGTTT    420                                                                 - - ATTGCAATTG ATGGCACAAG TTTAACAGTG GTGGATGTGT TTGACCAAGA AT -            #TATGCTTT    480                                                                 - - AATATTATGT TAGTTGCTTA CACTCAACAA AATGTGGTCA TTCCACTCAA AA -            #AAGTTGGC    540                                                                 - - CAAAAGGTTA ATTTAGAGGT TGATATTCTA GGAAAATATG TGGAAAGGCT CC -            #TAAGTAGT    600                                                                 - - AGTGGGGTTT TGGATCCTAC CAAATTCACA TAG       - #                  -      #        633                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  210 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Mature Spinach RS                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - Met Phe Thr Gly Ile Val Glu Glu Ile Gly Ar - #g Val Lys Gln Met        Gly                                                                              1               5  - #                 10 - #                 15             - - Tyr Gly Glu Asp Gly Gly Phe Gln Leu Lys Va - #l Val Gly Asp Ile Val                   20     - #             25     - #             30                  - - Leu Lys Asp Val Asn Leu Gly Asp Ser Ile Al - #a Val Asn Gly Thr Cys               35         - #         40         - #         45                      - - Leu Thr Val Thr Glu Phe Asp Thr Lys Ala Se - #r Glu Phe Thr Leu Gly           50             - #     55             - #     60                          - - Ile Ala Pro Glu Thr Leu Arg Lys Thr Ala Le - #u Met Asp Leu Glu Pro       65                 - # 70                 - # 75                 - # 80       - - Gly Ser Val Val Asn Leu Glu Arg Ala Leu Le - #u Pro Ser Thr Arg Met                       85 - #                 90 - #                 95              - - Gly Gly His Phe Val Gln Gly His Val Asp Gl - #y Thr Gly Glu Ile Val                  100      - #           105      - #           110                  - - Ser Leu Val Glu Glu Gly Asp Ser Leu Trp Va - #l Lys Ile Lys Thr Ser              115          - #       120          - #       125                      - - Pro Glu Ile Leu Arg Tyr Ile Val Pro Lys Gl - #y Phe Ile Ala Ile Asp          130              - #   135              - #   140                          - - Gly Thr Ser Leu Thr Val Val Asp Val Phe As - #p Gln Glu Leu Cys Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asn Ile Met Leu Val Ala Tyr Thr Gln Gln As - #n Val Val Ile Pro        Leu                                                                                             165  - #               170  - #               175             - - Lys Lys Val Gly Gln Lys Val Asn Leu Glu Va - #l Asp Ile Leu Gly Lys                  180      - #           185      - #           190                  - - Tyr Val Glu Arg Leu Leu Ser Ser Ser Gly Va - #l Leu Asp Pro Thr Lys              195          - #       200          - #       205                      - - Phe Thr                                                                      210                                                                        - - (2) INFORMATION FOR SEQ ID NO:9:                                          - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  627 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Mature arabidopsis RS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:                          - - GTGTTTACTG GAATCGTGGA GGAAATGGGT GAAGTCAAGG ACTTGGGAAT GG -             #CCGATCAC     60                                                                 - - GGAGGATTCG ACCTCAAAAT CGGAGCGAGA GTGGTGTTAG AGGACGTGAA GC -            #TCGGTGAC    120                                                                 - - AGTATCGCCG TGAACGGTAC TTGTTTAACG GTGACGGAGT TTAACGCAGA GG -            #AGTTCACA    180                                                                 - - GTAGGGTTAG CACCGGAGAC GCTGAGAAAA ACATCGTTGG AGGAGTTAAA GA -            #AAGGATCT    240                                                                 - - CCGGTGAATC TGGAGCGTGC GTTGCAGCCA GTGAGCAGGA TGGGTGGACA CG -            #TGGTTCAG    300                                                                 - - GGACACGTGG ATGGGACGGG AGTGATTGAA TCAATGGAGG TAGAGGGTGA TT -            #CTTTGTGG    360                                                                 - - GTGAAGGTGA AAGCTGACAA GGGTTTGTTG AAATACATTG TGCCTAAAGG AT -            #TTGTGGCT    420                                                                 - - GTTGATGGGA CTAGCTTGAC GGTTGTTGAT GTGTTTGATG AAGAGAGCTG TT -            #TCAATTTC    480                                                                 - - ATGATGATTG CTTATACGCA ACAGAATGTA GTGATTCCGA CTAAGAAGAT TG -            #GGCAGAAA    540                                                                 - - GTGAATCTTG AGGTTGATAT CATGGGGAAG TATGTTGAGA GGCTTCTCAC CA -            #GTGGTGGC    600                                                                 - - TTCTCCAAAG GAAAAGAAAA TATTTGA          - #                  - #                627                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  208 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Mature arabidposis RS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:                         - - Val Phe Thr Gly Ile Val Glu Glu Met Gly Gl - #u Val Lys Asp Leu Gly       1               5  - #                 10 - #                 15              - - Met Ala Asp His Gly Gly Phe Asp Leu Lys Il - #e Gly Ala Arg Val Val                   20     - #             25     - #             30                  - - Leu Glu Asp Val Lys Leu Gly Asp Ser Ile Al - #a Val Asn Gly Thr Cys               35         - #         40         - #         45                      - - Leu Thr Val Thr Glu Phe Asn Ala Glu Glu Ph - #e Thr Val Gly Leu Ala           50             - #     55             - #     60                          - - Pro Glu Thr Leu Arg Lys Thr Ser Leu Glu Gl - #u Leu Lys Lys Gly Ser      65                  - #70                  - #75                  - #80        - - Pro Val Asn Leu Glu Arg Ala Leu Gln Pro Va - #l Ser Arg Met Gly Gly                       85 - #                 90 - #                 95              - - His Val Val Gln Gly His Val Asp Gly Thr Gl - #y Val Ile Glu Ser Met                  100      - #           105      - #           110                  - - Glu Val Glu Gly Asp Ser Leu Trp Val Lys Va - #l Lys Ala Asp Lys Gly              115          - #       120          - #       125                      - - Leu Leu Lys Tyr Ile Val Pro Lys Gly Phe Va - #l Ala Val Asp Gly Thr          130              - #   135              - #   140                          - - Ser Leu Thr Val Val Asp Val Phe Asp Glu Gl - #u Ser Cys Phe Asn Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Met Met Ile Ala Tyr Thr Gln Gln Asn Val Va - #l Ile Pro Thr Lys        Lys                                                                                             165  - #               170  - #               175             - - Ile Gly Gln Lys Val Asn Leu Glu Val Asp Il - #e Met Gly Lys Tyr Val                  180      - #           185      - #           190                  - - Glu Arg Leu Leu Thr Ser Gly Gly Phe Ser Ly - #s Gly Lys Glu Asn Ile              195          - #       200          - #       205                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  645 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # M. grisea RS                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #11:                         - - ATGTTCACTG GTATAGTCGA GGAGATCGGA GTCGTGGCCG AGCTCAACCC GC -             #ACGATGCC     60                                                                 - - ACTGGAGGGA CGTCATTGAC CATCTCGCTC CCGACGGGCA GCAGCCTGCT CT -            #CGGATTGC    120                                                                 - - CACGACGGTG ATAGCATCGC CGTCAACGGT GTGTGCCTGA CCGTCACATC CT -            #TCACGCCG    180                                                                 - - ACGCAGTTCA CAGTCGGTGT TGCCCCGGAG ACGCTGCGCG TCACGGACCT GG -            #GCGACCTG    240                                                                 - - GTCAAGGACT CGCGCGTGAA CCTGGAGCGA GCCGTGCGGG CCGACACTCG CA -            #TGGGCGGT    300                                                                 - - CACTTTGTAC AGGGCCACGT CGACACGACC GCCACCATAG CCGACAAGCA GG -            #CAGATGGT    360                                                                 - - AACGCCGTCA CGATGCGGTT CAAGCCACGG GAGGGTAGCG ATGTGTTGAA GT -            #ACATCGTG    420                                                                 - - CGAAAGGGTT ATGTCGCATT GGACGGAACC AGCTTGACGG TTACTAAGGT CG -            #ACGACGCT    480                                                                 - - GCCGGGTGGT GGGAGGTCAT GCTCATCGTT TACACGCAGG AACGTGTGGT CC -            #TGGCGCAG    540                                                                 - - AAGAACGTTG GTGATACTGT CAATGTCGAG GTTGACGTCT TGGCCAAGTA TG -            #CTGAGAAG    600                                                                 - - AGTATGGCTG GATACTTGAG CTCTCTCAAC AAGAGTGACG CATAA   - #                     645                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  214 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # M. grisea RS                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #12:                         - - Met Phe Thr Gly Ile Val Glu Glu Ile Gly Va - #l Val Ala Glu Leu Asn       1               5  - #                 10 - #                 15              - - Pro His Asp Ala Thr Gly Gly Thr Ser Leu Th - #r Ile Ser Leu Pro Thr                   20     - #             25     - #             30                  - - Gly Ser Ser Leu Leu Ser Asp Cys His Asp Gl - #y Asp Ser Ile Ala Val               35         - #         40         - #         45                      - - Asn Gly Val Cys Leu Thr Val Thr Ser Phe Th - #r Pro Thr Gln Phe Thr           50             - #     55             - #     60                          - - Val Gly Val Ala Pro Glu Thr Leu Arg Val Th - #r Asp Leu Gly Asp Leu       65                 - # 70                 - # 75                 - # 80       - - Val Lys Asp Ser Arg Val Asn Leu Glu Arg Al - #a Val Arg Ala Asp Thr                       85 - #                 90 - #                 95              - - Arg Met Gly Gly His Phe Val Gln Gly His Va - #l Asp Thr Thr Ala Thr                  100      - #           105      - #           110                  - - Ile Ala Asp Lys Gln Ala Asp Gly Asn Ala Va - #l Thr Met Arg Phe Lys              115          - #       120          - #       125                      - - Pro Arg Glu Gly Ser Asp Val Leu Lys Tyr Il - #e Val Arg Lys Gly Tyr          130              - #   135              - #   140                          - - Val Ala Leu Asp Gly Thr Ser Leu Thr Val Th - #r Lys Val Asp Asp Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Gly Trp Trp Glu Val Met Leu Ile Val Ty - #r Thr Gln Glu Arg        Val                                                                                             165  - #               170  - #               175             - - Val Leu Ala Gln Lys Asn Val Gly Asp Thr Va - #l Asn Val Glu Val Asp                  180      - #           185      - #           190                  - - Val Leu Ala Lys Tyr Ala Glu Lys Ser Met Al - #a Gly Tyr Leu Ser Ser              195          - #       200          - #       205                      - - Leu Asn Lys Ser Asp Ala                                                      210                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  38 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli LS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #13:                         - - CGAAGGAAGA CCATGGCCAT TATTGAAGCT AACGTTGC      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  34 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' E. coli LS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #14:                         - - ATCTTACTGT CGACTTCAGG CCTTGATGGC TTTC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  35 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli RS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #15:                         - - ACTCATTTAC CATGGCTACG GGGATTGTAC AGGGC       - #                       - #       35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  35 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' primer E. coli RS                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #16:                         - - ATCTTACTGT CGACTTCAGG CTTCTGTGCC TGGTT       - #                       - #       35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli RS and LS primer             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #17:                         - - AACTAGATCA GCGGCCGCAG CCACGTTGTG TCTCAAA      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  36 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' E. coli LS and RS primer             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #18:                         - - GACAAACATA GCGGCCGCTG AGGTCTGCCT CGTGAA      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  36 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli LS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #19:                         - - AACTAGATCA GCGGCCGCGG TACGGTTATT CGTGGT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli LS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #20:                         - - GACAAACATA GCGGCCGCGT CGTATTTACC GGT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  35 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli RS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #21:                         - - AACTAGATCA GCGGCCGCAC CACTGCTGAA GTGGC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli RS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #22:                         - - GACAAACATA GCGGCCGCGA CCTGACATTA AGTGTCC      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  34 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' primer spinach RS                                   precursor                                                       - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #23:                         - - CTACTCATTT CATATGTTCA CTGGCATTGT TGAA       - #                  -     #        34                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' spinach RS precursor                                primer                                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #24:                         - - CATCTTACTG GATCCACTAT GTGAATTTGG TAGGATC      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  39 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' spinach LS precursor                                primer                                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #25:                         - - CTACTCATTT CATATGAACG AGCTTGAAGG TTATGTCAC      - #                      - #    39                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  38 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' primer spinach LS                                   precursor                                                       - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #26:                         - - CATCTTACTG GATCCATCAG GCCTTCAAAT GATGTTCG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  669 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # spinach LS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #27:                         - - ATGGCTTCAT TTGCAGCTTC TCAAACTTGT TTCCTGACAA CAAACCCCAC TT -             #GTTTAAAA     60                                                                 - - CCCAATTCCC CTCAAAAATC TTCCACATTT CTTCCATTTT CTGCCCCTCT TT -            #CTTCCTCG    120                                                                 - - TCATCTTTCC CTGGTTGTGG GTTGGTTCAT GTTGCATCAA ACAAGAAAAA TC -            #GTGCTTCG    180                                                                 - - TTTGTAGTGA CCAATGCTGT TAGGGAGCTT GAAGGTTATG TCACTAAAGC CC -            #AGAGTTTC    240                                                                 - - AGATTTGCCA TTGTTGTGGC TAGGTTCAAC GAATTTGTGA CAAGACGACT AA -            #TGGAAGGA    300                                                                 - - GCTCTTGACA CTTTTAAGAA ATACTCTGTC AATGAAGATA TTGATGTTGT TT -            #GGGTTCCT    360                                                                 - - GGTGCTTATG AGCTAGGTGT TACTGCACAA GCACTTGGGA AATCAGGAAA AT -            #ATCATGCT    420                                                                 - - ATTGTTTGTC TTGGAGCTGT GGTAAAAGGT GATACTTCAC ACTATGATGC TG -            #TCGTTAAT    480                                                                 - - TCTGCTTCCT CTGGAGTACT GTCAGCTGGA TTAAATTCAG GAGTACCTTG TG -            #TCTTTGGT    540                                                                 - - GTCCTTACCT GTGATAACAT GGATCAGGCC ATAAATCGAG CTGGCGGGAA AG -            #CGGGTAAT    600                                                                 - - AAAGGAGCCG AGTCAGCGCT AACAGCTATT GAAATGGCTT CGCTTTTCGA AC -            #ATCATTTG    660                                                                 - - AAGGCCTAA                - #                  - #                      - #        669                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  222 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # spinach LS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #28:                         - - Met Ala Ser Phe Ala Ala Ser Gln Thr Cys Ph - #e Leu Thr Thr Asn Pro       1               5  - #                 10 - #                 15              - - Thr Cys Leu Lys Pro Asn Ser Pro Gln Lys Se - #r Ser Thr Phe Leu Pro                   20     - #             25     - #             30                  - - Phe Ser Ala Pro Leu Ser Ser Ser Ser Ser Ph - #e Pro Gly Cys Gly Leu               35         - #         40         - #         45                      - - Val His Val Ala Ser Asn Lys Lys Asn Arg Al - #a Ser Phe Val Val Thr           50             - #     55             - #     60                          - - Asn Ala Val Arg Glu Leu Glu Gly Tyr Val Th - #r Lys Ala Gln Ser Phe       65                 - # 70                 - # 75                 - # 80       - - Arg Phe Ala Ile Val Val Ala Arg Phe Asn Gl - #u Phe Val Thr Arg Arg                       85 - #                 90 - #                 95              - - Leu Met Glu Gly Ala Leu Asp Thr Phe Lys Ly - #s Tyr Ser Val Asn Glu                  100      - #           105      - #           110                  - - Asp Ile Asp Val Val Trp Val Pro Gly Ala Ty - #r Glu Leu Gly Val Thr              115          - #       120          - #       125                      - - Ala Gln Ala Leu Gly Lys Ser Gly Lys Tyr Hi - #s Ala Ile Val Cys Leu          130              - #   135              - #   140                          - - Gly Ala Val Val Lys Gly Asp Thr Ser His Ty - #r Asp Ala Val Val Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Ala Ser Ser Gly Val Leu Ser Ala Gly Le - #u Asn Ser Gly Val        Pro                                                                                             165  - #               170  - #               175             - - Cys Val Phe Gly Val Leu Thr Cys Asp Asn Me - #t Asp Gln Ala Ile Asn                  180      - #           185      - #           190                  - - Arg Ala Gly Gly Lys Ala Gly Asn Lys Gly Al - #a Glu Ser Ala Leu Thr              195          - #       200          - #       205                      - - Ala Ile Glu Met Ala Ser Leu Phe Glu His Hi - #s Leu Lys Ala                  210              - #   215              - #   220                          - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  678 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # tobacco LS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #29:                         - - TTCGCTTTCG GACAGTGCAA TCTTCTACCT CGTACAACAA CTGTAAATCC CA -             #CACAACTG     60                                                                 - - CACTCTCCTC TTTACTCTTT GTCTTTGCCT TTCCACAGAC AAAGCATAAC CT -            #CTTCACCT    120                                                                 - - GCACTATCAT TCACCCAATC TCAAGGTTTA GGGTCTGCAA TTGAGAGACA TT -            #GCGACCGG    180                                                                 - - TCGGATCTGT TTCAAACATG TGCTGTTCGT CAGTTGACTG GTTCTGTTAC CT -            #CTGCCAAA    240                                                                 - - GGCCATCGCT TTGCTGTTGT GGTTGCACGT TTCAATGATC TTATCACCAA GA -            #AGCTTTTG    300                                                                 - - GAGGGAGCTT TGGACACTTT CAAAAATTAC TCTGTTAGAG AGGAAGATAT TG -            #ATGTCGTG    360                                                                 - - TGGGTTCCTG GCTGTTTTGA AATCGGTGTG GTTGCGCAAC AGCTTGGAAA GT -            #CGCAGAAA    420                                                                 - - TATCAAGCAA TACTCTGTAT TGGGGCTGTG ATTAGAGGTG ATACGTCTCA CT -            #ATGATGCC    480                                                                 - - GTCGTTAATG CTGCCACATC CGGAGTACTT TCAGCAGGTC TAAATTCTGG TA -            #CTCCTTGC    540                                                                 - - ATATTTGGTG TTTTGACATG TGATACCTTG GAGCAGGCTT TCAATCGTGT CG -            #GTGGGAAG    600                                                                 - - GCTGGGAATA AAGGTGCCGA AACAGCGTTG ACAGCTATTG AGATGGCGTC TT -            #TGTTTGAA    660                                                                 - - CACCACTTAA AGGCTTAA             - #                  - #                      - # 678                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  225 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # tobacco LS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #30:                         - - Phe Ala Phe Gly Gln Cys Asn Leu Leu Pro Ar - #g Thr Thr Thr Val Asn       1               5  - #                 10 - #                 15              - - Pro Thr Gln Leu His Ser Pro Leu Tyr Ser Le - #u Ser Leu Pro Phe His                   20     - #             25     - #             30                  - - Arg Gln Ser Ile Thr Ser Ser Pro Ala Leu Se - #r Phe Thr Gln Ser Gln               35         - #         40         - #         45                      - - Gly Leu Gly Ser Ala Ile Glu Arg His Cys As - #p Arg Ser Asp Leu Phe           50             - #     55             - #     60                          - - Gln Thr Cys Ala Val Arg Gln Leu Thr Gly Se - #r Val Thr Ser Ala Lys      65                  - #70                  - #75                  - #80        - - Gly His Arg Phe Ala Val Val Val Ala Arg Ph - #e Asn Asp Leu Ile Thr                       85 - #                 90 - #                 95              - - Lys Lys Leu Leu Glu Gly Ala Leu Asp Thr Ph - #e Lys Asn Tyr Ser Val                  100      - #           105      - #           110                  - - Arg Glu Glu Asp Ile Asp Val Val Trp Val Pr - #o Gly Cys Phe Glu Ile              115          - #       120          - #       125                      - - Gly Val Val Ala Gln Gln Leu Gly Lys Ser Gl - #n Lys Tyr Gln Ala Ile          130              - #   135              - #   140                          - - Leu Cys Ile Gly Ala Val Ile Arg Gly Asp Th - #r Ser His Tyr Asp Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Val Asn Ala Ala Thr Ser Gly Val Leu Se - #r Ala Gly Leu Asn        Ser                                                                                             165  - #               170  - #               175             - - Gly Thr Pro Cys Ile Phe Gly Val Leu Thr Cy - #s Asp Thr Leu Glu Gln                  180      - #           185      - #           190                  - - Ala Phe Asn Arg Val Gly Gly Lys Ala Gly As - #n Lys Gly Ala Glu Thr              195          - #       200          - #       205                      - - Ala Leu Thr Ala Ile Glu Met Ala Ser Leu Ph - #e Glu His His Leu Lys          210              - #   215              - #   220                          - - Ala                                                                      225                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  684 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # arabidopsis LS precursor                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #31:                         - - ATGAAGTCAT TAGCTTCGCC GCCGTGTCTC CGCCTGATAC CGACGGCACA CC -             #GTCAGCTC     60                                                                 - - AATTCGCGTC AATCTTCCTC CGCCTGTTAT ATACACGGTG GCTCTTCTGT GA -            #ACAAATCC    120                                                                 - - AATAATCTCT CATTCTCCTC ATCCACATCC GGATTTGCGT CACCACTAGC TG -            #TAGAGAAG    180                                                                 - - GAATTACGCT CTTCATTCGT ACAGACGGCT GCTGTTCGCC ATGTTACGGG GT -            #CTCTTATC    240                                                                 - - AGAGGCGAAG GTCTTAGATT CGCCATCGTG GTAGCTCGTT TCAATGAGGT TG -            #TGACTAAG    300                                                                 - - TTGCTTTTGG AAGGAGCGAT TGAGACTTTC AAGAAGTATT CAGTCAGAGA AG -            #AAGACATT    360                                                                 - - GAAGTTATTT GGGTTCCTGG CAGCTTTGAA ATTGGTGTTG TTGCACAAAA TC -            #TTGGGAAA    420                                                                 - - TCGGGAAAAT TTCATGCTGT TTTATGTATC GGCGCTGTGA TAAGAGGAGA TA -            #CCACACAT    480                                                                 - - TATGATGCTG TTGCCAACTC TGCTGCGTCT GGAGTACTTT CTGCTAGCAT AA -            #ATTCAGGC    540                                                                 - - GTTCCATGCA TATTTGGTGT ACTGACTTGC GAGGACATGG ATCAGGCTCT GA -            #ATCGATCT    600                                                                 - - GGTGGCAAAG CCGGCAATAA GGGAGCTGAA ACTGCTTTGA CGGCGCTCGA AA -            #TGGCGTCG    660                                                                 - - TTGTTTGAGC ACCACCTGAA ATAG          - #                  - #                   684                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  227 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # arabidopsis LS precursor                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #32:                         - - Met Lys Ser Leu Ala Ser Pro Pro Cys Leu Ar - #g Leu Ile Pro Thr Ala       1               5  - #                 10 - #                 15              - - His Arg Gln Leu Asn Ser Arg Gln Ser Ser Se - #r Ala Cys Tyr Ile His                   20     - #             25     - #             30                  - - Gly Gly Ser Ser Val Asn Lys Ser Asn Asn Le - #u Ser Phe Ser Ser Ser               35         - #         40         - #         45                      - - Thr Ser Gly Phe Ala Ser Pro Leu Ala Val Gl - #u Lys Glu Leu Arg Ser           50             - #     55             - #     60                          - - Ser Phe Val Gln Thr Ala Ala Val Arg His Va - #l Thr Gly Ser Leu Ile      65                  - #70                  - #75                  - #80        - - Arg Gly Glu Gly Leu Arg Phe Ala Ile Val Va - #l Ala Arg Phe Asn Glu                       85 - #                 90 - #                 95              - - Val Val Thr Lys Leu Leu Leu Glu Gly Ala Il - #e Glu Thr Phe Lys Lys                  100      - #           105      - #           110                  - - Tyr Ser Val Arg Glu Glu Asp Ile Glu Val Il - #e Trp Val Pro Gly Ser              115          - #       120          - #       125                      - - Phe Glu Ile Gly Val Val Ala Gln Asn Leu Gl - #y Lys Ser Gly Lys Phe          130              - #   135              - #   140                          - - His Ala Val Leu Cys Ile Gly Ala Val Ile Ar - #g Gly Asp Thr Thr His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Asp Ala Val Ala Asn Ser Ala Ala Ser Gl - #y Val Leu Ser Ala        Ser                                                                                             165  - #               170  - #               175             - - Ile Asn Ser Gly Val Pro Cys Ile Phe Gly Va - #l Leu Thr Cys Glu Asp                  180      - #           185      - #           190                  - - Met Asp Gln Ala Leu Asn Arg Ser Gly Gly Ly - #s Ala Gly Asn Lys Gly              195          - #       200          - #       205                      - - Ala Glu Thr Ala Leu Thr Ala Leu Glu Met Al - #a Ser Leu Phe Glu His          210              - #   215              - #   220                          - - His Leu Lys                                                              225                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  840 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Spinach RS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #33:                         - - ATGGCACTTT CAACTTCACT CTCTTTAGTA TCTCCCAAAC TCTCTCAACA AA -             #ATCTCACA     60                                                                 - - TTTTGCACCT TCAACAACCA ACCCTCCTCT TTAAATGGGC ATATCAAATT CA -            #ATCCAAAC    120                                                                 - - CTCAGAAACT CAGTCTCTAA ACTCTTTATC ACCACCCAAA ACACCCGATT CC -            #TAAAATTT    180                                                                 - - CGGTACGTAA GGAATCAAAT AAACTCCATG TTCACTGGCA TTGTTGAAGA GA -            #TTGGCCGA    240                                                                 - - GTTAAGCAAA TGGGTTATGG CGAAGACGGT GGATTTCAGC TTAAAGTTGT AG -            #GAGACATT    300                                                                 - - GTCCTAAAAG ATGTCAATCT TGGTGACAGT ATCGCAGTTA ATGGTACATG TC -            #TAACTGTG    360                                                                 - - ACGGAATTTG ACACTAAAGC GTCCGAATTT ACTCTTGGGA TAGCGCCTGA GA -            #CGCTTAGG    420                                                                 - - AAGACGGCAT TGATGGATCT CGAACCAGGG TCAGTTGTTA ATTTAGAAAG AG -            #CCCTTTTG    480                                                                 - - CCTTCTACAC GGATGGGTGG TCACTTTGTC CAGGGACATG TTGATGGGAC AG -            #GAGAAATT    540                                                                 - - GTATCACTAG TTGAAGAAGG TGATTCTTTG TGGGTCAAGA TAAAAACAAG CC -            #CAGAAATA    600                                                                 - - CTGAGATACA TTGTACCAAA AGGGTTTATT GCAATTGATG GCACAAGTTT AA -            #CAGTGGTG    660                                                                 - - GATGTGTTTG ACCAAGAATT ATGCTTTAAT ATTATGTTAG TTGCTTACAC TC -            #AACAAAAT    720                                                                 - - GTGGTCATTC CACTCAAAAA AGTTGGCCAA AAGGTTAATT TAGAGGTTGA TA -            #TTCTAGGA    780                                                                 - - AAATATGTGG AAAGGCTCCT AAGTAGTAGT GGGGTTTTGG ATCCTACCAA AT -            #TCACATAG    840                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  279 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Spinach RS precursor                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #34:                         - - Met Ala Leu Ser Thr Ser Leu Ser Leu Val Se - #r Pro Lys Leu Ser        Gln                                                                              1               5  - #                 10 - #                 15             - - Gln Asn Leu Thr Phe Cys Thr Phe Asn Asn Gl - #n Pro Ser Ser Leu Asn                   20     - #             25     - #             30                  - - Gly His Ile Lys Phe Asn Pro Asn Leu Arg As - #n Ser Val Ser Lys Leu               35         - #         40         - #         45                      - - Phe Ile Thr Thr Gln Asn Thr Arg Phe Leu Ly - #s Phe Arg Tyr Val Arg           50             - #     55             - #     60                          - - Asn Gln Ile Asn Ser Met Phe Thr Gly Ile Va - #l Glu Glu Ile Gly Arg      65                  - #70                  - #75                  - #80        - - Val Lys Gln Met Gly Tyr Gly Glu Asp Gly Gl - #y Phe Gln Leu Lys Val                       85 - #                 90 - #                 95              - - Val Gly Asp Ile Val Leu Lys Asp Val Asn Le - #u Gly Asp Ser Ile Ala                  100      - #           105      - #           110                  - - Val Asn Gly Thr Cys Leu Thr Val Thr Glu Ph - #e Asp Thr Lys Ala Ser              115          - #       120          - #       125                      - - Glu Phe Thr Leu Gly Ile Ala Pro Glu Thr Le - #u Arg Lys Thr Ala Leu          130              - #   135              - #   140                          - - Met Asp Leu Glu Pro Gly Ser Val Val Asn Le - #u Glu Arg Ala Leu Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Ser Thr Arg Met Gly Gly His Phe Val Gl - #n Gly His Val Asp        Gly                                                                                             165  - #               170  - #               175             - - Thr Gly Glu Ile Val Ser Leu Val Glu Glu Gl - #y Asp Ser Leu Trp Val                  180      - #           185      - #           190                  - - Lys Ile Lys Thr Ser Pro Glu Ile Leu Arg Ty - #r Ile Val Pro Lys Gly              195          - #       200          - #       205                      - - Phe Ile Ala Ile Asp Gly Thr Ser Leu Thr Va - #l Val Asp Val Phe Asp          210              - #   215              - #   220                          - - Gln Glu Leu Cys Phe Asn Ile Met Leu Val Al - #a Tyr Thr Gln Gln Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Val Ile Pro Leu Lys Lys Val Gly Gln Ly - #s Val Asn Leu Glu        Val                                                                                             245  - #               250  - #               255             - - Asp Ile Leu Gly Lys Tyr Val Glu Arg Leu Le - #u Ser Ser Ser Gly Val                  260      - #           265      - #           270                  - - Leu Asp Pro Thr Lys Phe Thr                                                      275                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  816 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # arabidopsis RS precursor                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #35:                         - - ATGATGGCGG CTCGTACTCA TTGTATCAAC CTTATCCCCA AAGTATGTCT TC -             #CACAATCC     60                                                                 - - TTCAGAACTG GAGAATCAGT GACTAATCTC AGATTTGATT GCGTCTCTAA GT -            #CATCGAAG    120                                                                 - - CTTTCTCTCA AGACATCATG TGGAAGATCA AGAACGCATC ACCGGAGGCA AA -            #ATCTCAGC    180                                                                 - - ATCCGGTCCG TGTTTACTGG AATCGTGGAG GAAATGGGTG AAGTCAAGGA CT -            #TGGGAATG    240                                                                 - - GCCGATCACG GAGGATTCGA CCTCAAAATC GGAGCGAGAG TGGTGTTAGA GG -            #ACGTGAAG    300                                                                 - - CTCGGTGACA GTATCGCCGT GAACGGTACT TGTTTAACGG TGACGGAGTT TA -            #ACGCAGAG    360                                                                 - - GAGTTCACAG TAGGGTTAGC ACCGGAGACG CTGAGAAAAA CATCGTTGGA GG -            #AGTTAAAG    420                                                                 - - AAAGGATCTC CGGTGAATCT GGAGCGTGCG TTGCAGCCAG TGAGCAGGAT GG -            #GTGGACAC    480                                                                 - - GTGGTTCAGG GACACGTGGA TGGGACGGGA GTGATTGAAT CAATGGAGGT AG -            #AGGGTGAT    540                                                                 - - TCTTTGTGGG TGAAGGTGAA AGCTGACAAG GGTTTGTTGA AATACATTGT GC -            #CTAAAGGA    600                                                                 - - TTTGTGGCTG TTGATGGGAC TAGCTTGACG GTTGTTGATG TGTTTGATGA AG -            #AGAGCTGT    660                                                                 - - TTCAATTTCA TGATGATTGC TTATACGCAA CAGAATGTAG TGATTCCGAC TA -            #AGAAGATT    720                                                                 - - GGGCAGAAAG TGAATCTTGA GGTTGATATC ATGGGGAAGT ATGTTGAGAG GC -            #TTCTCACC    780                                                                 - - AGTGGTGGCT TCTCCAAAGG AAAAGAAAAT ATTTGA      - #                       - #      816                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  271 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # arabidopsis RS precursor                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #36:                         - - Met Met Ala Ala Arg Thr His Cys Ile Asn Le - #u Ile Pro Lys Val        Cys                                                                              1               5  - #                 10 - #                 15             - - Leu Pro Gln Ser Phe Arg Thr Gly Glu Ser Va - #l Thr Asn Leu Arg Phe                   20     - #             25     - #             30                  - - Asp Cys Val Ser Lys Ser Ser Lys Leu Ser Le - #u Lys Thr Ser Cys Gly               35         - #         40         - #         45                      - - Arg Ser Arg Thr His His Arg Arg Gln Asn Le - #u Ser Ile Arg Ser Val           50             - #     55             - #     60                          - - Phe Thr Gly Ile Val Glu Glu Met Gly Glu Va - #l Lys Asp Leu Gly Met      65                  - #70                  - #75                  - #80        - - Ala Asp His Gly Gly Phe Asp Leu Lys Ile Gl - #y Ala Arg Val Val Leu                       85 - #                 90 - #                 95              - - Glu Asp Val Lys Leu Gly Asp Ser Ile Ala Va - #l Asn Gly Thr Cys Leu                  100      - #           105      - #           110                  - - Thr Val Thr Glu Phe Asn Ala Glu Glu Phe Th - #r Val Gly Leu Ala Pro              115          - #       120          - #       125                      - - Glu Thr Leu Arg Lys Thr Ser Leu Glu Glu Le - #u Lys Lys Gly Ser Pro          130              - #   135              - #   140                          - - Val Asn Leu Glu Arg Ala Leu Gln Pro Val Se - #r Arg Met Gly Gly His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Val Gln Gly His Val Asp Gly Thr Gly Va - #l Ile Glu Ser Met        Glu                                                                                             165  - #               170  - #               175             - - Val Glu Gly Asp Ser Leu Trp Val Lys Val Ly - #s Ala Asp Lys Gly Leu                  180      - #           185      - #           190                  - - Leu Lys Tyr Ile Val Pro Lys Gly Phe Val Al - #a Val Asp Gly Thr Ser              195          - #       200          - #       205                      - - Leu Thr Val Val Asp Val Phe Asp Glu Glu Se - #r Cys Phe Asn Phe Met          210              - #   215              - #   220                          - - Met Ile Ala Tyr Thr Gln Gln Asn Val Val Il - #e Pro Thr Lys Lys Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Gln Lys Val Asn Leu Glu Val Asp Ile Me - #t Gly Lys Tyr Val        Glu                                                                                             245  - #               250  - #               255             - - Arg Leu Leu Thr Ser Gly Gly Phe Ser Lys Gl - #y Lys Glu Asn Ile                      260      - #           265      - #           270                  - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  603 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # M. grisea LS                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #37:                         - - ATGCACACCA AAGGCCCGAC CCCGCAGCAG CACGACGGCT CCGCCCTGCG CA -             #TCGGCATC     60                                                                 - - GTGCACGCGC GCTGGAACGA GACCATCATC GAGCCGCTTC TGGCCGGCAC AA -            #AAGCCAAG    120                                                                 - - CTGCTGGCCT GCGGCGTCAA GGAGTCCAAC ATAGTCGTGC AGAGCGTTCC GG -            #GGTCGTGG    180                                                                 - - GAGCTGCCAA TAGCCGTGCA GAGGCTCTAC TCCGCATCCC AGCTCCAAAC CC -            #CAAGCTCC    240                                                                 - - GGCCCATCTC TGTCGGCCGG CGACCTGCTC GGCTCCTCGA CCACAGATCT TA -            #CCGCGCTC    300                                                                 - - CCGACCACCA CTGCCTCATC CACCGGCCCC TTTGACGCCC TCATCGCCAT CG -            #GCGTGCTA    360                                                                 - - ATCAAGGGCG AGACGATGCA CTTTGAGTAC ATTGCCGATT CGGTCTCGCA CG -            #GCCTGATG    420                                                                 - - CGCGTACAGC TCGACACGGG CGTCCCAGTT ATCTTCGGCG TCCTAACAGT CC -            #TGACCGAC    480                                                                 - - GACCAGGCCA AGGCTCGTGC CGGCGTCATC GAGGGCAGCC ACAACCACGG CG -            #AGGACTGG    540                                                                 - - GGCCTGGCCG CCGTTGAGAT GGGTGTGCGC AGGAGGGATT GGGCTGCCGG GA -            #AGACCGAG    600                                                                 - - TGA                  - #                  - #                  - #                603                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  200 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # M. grisea LS                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #38:                         - - Met His Thr Lys Gly Pro Thr Pro Gln Gln Hi - #s Asp Gly Ser Ala Leu       1               5  - #                 10 - #                 15              - - Arg Ile Gly Ile Val His Ala Arg Trp Asn Gl - #u Thr Ile Ile Glu Pro                   20     - #             25     - #             30                  - - Leu Leu Ala Gly Thr Lys Ala Lys Leu Leu Al - #a Cys Gly Val Lys Glu               35         - #         40         - #         45                      - - Ser Asn Ile Val Val Gln Ser Val Pro Gly Se - #r Trp Glu Leu Pro Ile           50             - #     55             - #     60                          - - Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Le - #u Gln Thr Pro Ser Ser      65                  - #70                  - #75                  - #80        - - Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gl - #y Ser Ser Thr Thr Asp                       85 - #                 90 - #                 95              - - Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Se - #r Thr Gly Pro Phe Asp                  100      - #           105      - #           110                  - - Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gl - #y Glu Thr Met His Phe              115          - #       120          - #       125                      - - Glu Tyr Ile Ala Asp Ser Val Ser His Gly Le - #u Met Arg Val Gln Leu          130              - #   135              - #   140                          - - Asp Thr Gly Val Pro Val Ile Phe Gly Val Le - #u Thr Val Leu Thr Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Gl - #u Gly Ser His Asn        His                                                                                             165  - #               170  - #               175             - - Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Me - #t Gly Val Arg Arg Arg                  180      - #           185      - #           190                  - - Asp Trp Ala Ala Gly Lys Thr Glu                                                  195          - #       200                                             - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  9 amino - # acids                                                (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  peptide                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #39:                         - - Ala Ser Leu Phe Glu His His Leu Lys                                      1               5                                                            __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid fragment selected fromthe group consisting of:(a) an isolated nucleic acid fragment encodingthe amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:10; (b) anisolated nucleic acid fragment encoding a polypeptide having riboflavinsynthase(RS)enzyme activity, wherein said isolated nucleic acid fragmenthybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C.; (c) an isolated nucleic acid fragmentencoding a polypeptide having riboflavin synthase (RS) enzyme activitywherein said encoded polypeptide has at least 70% identity with theamino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO: 10; and (d)an isolated nucleic acid fragment that is completely complementary tothe full length of (a), (b) or (c).
 2. The isolated nucleic acidfragment of claim 1 selected from the group consisting of SEQ ID NO:7and SEQ ID NO:9.
 3. The isolated nucleic acid fragment of claim 1encoding a plant riboflavin synthase enzyme, wherein the plant isspinach or arabidopsis.
 4. A chimeric gene comprising the isolatednucleic acid fragment of claim 1 operably linked to suitable regulatorysequences.
 5. A transformed host cell comprising a host cell and thechimeric gene of claim
 4. 6. The transformed host cell of claim 5wherein the host cell is a plant cell.
 7. The transformed host cell ofclaim 5 wherein the host cell is E. coli.
 8. A method of altering thelevel of expression of a plant riboflavin synthase enzyme in a host cellcomprising:(a) transforming a host cell with the chimeric gene of claim4; and (b) growing the transformed host cell of step (a) underconditions that are suitable for expression of the chimericgene,resulting in production of altered levels of a plant riboflavinsynthase enzyme in the transformed host cell relative to expressionlevels of an untransformed host cell.
 9. A method of obtaining a nucleicacid fragment encoding a plant riboflavin synthase enzyme comprising:(a)probing a cDNA or genomic library with the nucleic acid fragment ofclaim 1; (b) identifying a DNA clone that hybridizes with the nucleicacid fragment of claim 1 under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C.; (c) sequencing the cDNA or genomic fragmentthat comprises the clone identified in step (b),wherein the sequencedcDNA or genomic fragment encodes a plant riboflavin synthase enzyme. 10.A nucleic acid fragment encoding an enzymatically active plantriboflavin synthase enzyme obtained by the method comprising:(a) probinga cDNA or genomic library with the nucleic acid fragment set forth inSEQ ID NO:7 or SEQ ID NO:9; (b) identifying a DNA clone that hybridizeswith the nucleic acid fragment set forth in SEQ ID NO:7 or SEQ ID NO:9under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C.;and (c) sequencing the cDNA or genomic fragment that comprises the cloneidentified in step (b),wherein the sequenced cDNA or genomic fragmentencodes a plant riboflavin synthase enzyme.
 11. A nucleic acid fragmentencoding an enzymatically active plant riboflavin synthase enzymeobtained by the method comprising:(a) probing a cDNA or genomic librarywith a nucleic acid fragment encoding the amino acid sequence set forthin SEQ ID NO:8 or SEQ ID NO:10; (b) identifying a DNA clone thathybridizes with a nucleic acid fragment encoding the amino acid sequenceset forth in SEQ ID NO:8 or SEQ ID NO:10 under the followinghybridization conditions: 0.1×SSC, 0.1% SDS, 65° C.; and (c) sequencingthe cDNA or genomic fragment that comprises the clone identified in step(b),wherein the sequenced cDNA or genomic fragment encodes a plantriboflavin synthase enzyme.